Abstract

   Cancer cells rely on mitochondria for their bioenergetic supply and
   macromolecule synthesis. Central to mitochondrial function is the
   regulation of mitochondrial protein synthesis, which primarily depends
   on the cytoplasmic translation of nuclear-encoded mitochondrial mRNAs
   whose protein products are imported into mitochondria. Despite the
   growing evidence that mitochondrial protein synthesis contributes to
   the onset and progression of cancer, and can thus offer new
   opportunities for cancer therapy, knowledge of the underlying molecular
   mechanisms remains limited. Here, we show that RNA G-quadruplexes
   (RG4s) regulate mitochondrial function by modulating cytoplasmic mRNA
   translation of nuclear-encoded mitochondrial proteins. Our data support
   a model whereby the RG4 folding dynamics, under the control of
   oncogenic signaling and modulated by small molecule ligands or
   RG4-binding proteins, modifies mitochondria-localized cytoplasmic
   protein synthesis. Ultimately, this impairs mitochondrial functions,
   affecting energy metabolism and consequently cancer cell proliferation.

   Subject terms: Molecular biology, Translation
     __________________________________________________________________

   Cancer cells rely on mitochondria for energy and macromolecule
   synthesis. Here, the authors show that RNA G-quadruplex dynamics,
   driven by oncogenic signals, regulate the translation of
   nuclear-encoded mitochondrial mRNAs, affecting mitochondrial function
   and promoting cancer cell growth.

Introduction

   Contrary to conventional wisdom^[48]1, accumulating evidence supports
   the notion that mitochondrial metabolism is active in cancer cells and
   required for tumor growth, as it fuels the bioenergetic and
   biosynthetic needs of cancer cells^[49]2. Metabolic reprogramming with
   increased dependence on mitochondrial functions may increase during
   tumor progression^[50]3 and in response to therapies^[51]4,[52]5.
   Central to the mitochondrial function is the regulation of
   mitochondrial protein synthesis, which relies primarily on the
   cytoplasmic translation of nuclear-encoded mitochondrial mRNAs whose
   protein products are then imported into mitochondria^[53]6. The
   majority of these proteins are directly or indirectly involved in the
   mitochondrial translation of 13 mitochondrial-encoded membrane
   proteins. These proteins are all components of the respiratory chain,
   which generates the majority of cellular ATP via oxidative
   phosphorylation (OXPHOS). Recent data have shown that most
   mitochondrial protein-coding mRNAs are enriched at the mitochondrial
   outer membrane (OMM) in a translation-dependent manner and translated
   by mitochondria-associated cytoplasmic ribosomes, supporting the notion
   of localized translation at the OMM regulating the influx of
   mitochondrial proteins^[54]7. Despite the growing awareness that
   mitochondrial protein synthesis contributes to the onset and
   progression of cancer and thus offers new opportunities for cancer
   therapy^[55]8–[56]10, knowledge about the underlying molecular
   mechanisms remains limited.

   mRNA translation is the most energetically consuming process in the
   cell and its deregulation is widely recognized as a cancer
   hallmark^[57]11,[58]12. Current knowledge points towards the concept of
   mRNA translational reprogramming that selectively controls subgroups of
   mRNAs (also called “regulons”^[59]13) involved in defined cancer
   pathways^[60]14. Leveraging this concept to target specific oncogenic
   pathways for therapeutic purposes is currently limited by the lack of
   in-depth knowledge of the molecular determinants, i.e. RNA
   elements/structures and RNA-binding proteins (RBPs), as well as their
   mode of action and regulation, which, according to recent data, may
   involve specific localized factories^[61]15. RNA G-quadruplexes (G4),
   hereafter referred to as RG4s, are non-canonical structures consisting
   of G-rich sequences that form stacked tetrads of guanines (G-quartets)
   held together by Hoogsteen hydrogen bonds (for recent reviews, see
   Dumas et al. and Varshney et al.^[62]16,[63]17). The pervasive nature
   of RG4s has been demonstrated by surveying the extent of their folding
   both in vitro and in cellulo using sequencing-based
   methods^[64]18,[65]19 and optical imaging via immunodetection with
   G4-specific antibodies (including BG4^[66]20) or molecular probes
   (including biomimetic fluorescent probes^[67]21). The
   transcriptome-wide analysis of RG4 formation in living cells indicate
   that these are transient structures whose folding is regulated by RBPs
   and helicases^[68]18,[69]22, many of which have been identified by
   large-scale proteomic studies^[70]23–[71]31. Although RG4s are
   increasingly recognized as important in the translational regulation of
   cancer protein-coding mRNAs, their ability to target mRNA regulons was
   shown only by few studies^[72]26. Recent data highlighting the role of
   cytoplasmic translation in mitochondrial protein synthesis, suggested a
   potential role for RG4s in cytoplasmic translation of nuclear-encoded
   mRNAs^[73]32. This is consistent with the emerging view that G4s play
   an important role in mitochondria^[74]16,[75]33. Supporting this
   notion, G4s are found in mitochondria^[76]34–[77]36 where they function
   as modulators of genome replication, transcription^[78]37, and RNA
   stability^[79]38. Although this indicates a role for DNA/RNA G4s in
   mitochondrial biogenesis and mitochondrial DNA (mtDNA) gene expression,
   the possibility that RG4s affect mitochondrial functions by regulating
   cytoplasmic expression of mitochondrial proteins remains yet
   unconfirmed.

   Here we show that cytosolic RG4s regulate mitochondrial function by
   controlling cytoplasmic translation of nuclear-encoded mitochondrial
   mRNAs at the outer mitochondrial membrane. This function, which is
   mediated by their interaction with the RNA-binding protein hnRNP U and
   is controlled by mTOR oncogene signaling, results in altered
   mitochondrial functions affecting energy metabolism and therefore
   cancer cell proliferation.

Results

RG4s affect mitochondrial gene expression and function

   To investigate the link between RG4s and mitochondria, we first
   performed a functional enrichment analysis using transcriptome-wide
   datasets of in silico predicted^[80]39 or in vitro (HeLa or HEK293T
   cells) identified RG4s^[81]18,[82]19. This analysis revealed that
   several mitochondria-related terms were significantly enriched in
   RG4-containing mRNAs (Supplementary Fig. [83]1a). Consistent with this
   and with the notion that RG4s modulate gene expression^[84]16, we found
   that mitochondrial proteins encoded by RG4-containing mRNAs, including
   factors of the respiratory chain complex, were significantly enriched
   among differentially expressed proteins after treatment of HEK293T or
   HeLa cells with the RG4 ligand carboxypyridostatin (cPDS, a chemical
   derivative of PDS)^[85]40 (Supplementary Fig. [86]1b). These results,
   together with previous data highlighting oxidative phosphorylation as
   the most significantly enriched pathway among the proteins
   differentially expressed in HeLa cells treated with the G4 ligand
   20A^[87]41, suggest that RG4s may be involved in the regulation of
   mitochondrial gene expression and respiratory function in a cell type-
   and ligand-independent manner. To address these possibilities, we
   reasoned that RG4s regulating mitochondrial mRNA expression should be
   found in close proximity to mitochondria. Previous works using
   fluorescent G4 ligands or the BG4 antibody^[88]34–[89]36 mapped G4s to
   the mitochondrial genome. This result is consistent with the presence
   of G-rich sequences in mitochondrial DNA, but due to their uneven
   distribution between the two strands, the resulting transcriptome will
   have high or low G content. Indeed, in humans, the heavy DNA strand is
   G-rich and serves as a template for the transcription of most
   mitochondria-encoded genes, giving rise to RNAs (mRNAs, rRNAs, tRNAs)
   with low G-content. The light DNA strand is instead G-poor and mostly
   transcribes noncoding RNAs, which form RG4 structures and are degraded
   by the degradosome^[90]38. Based on these notions, it is therefore
   expected that mitochondria form DNA G4s but very little RNA G4s. In
   agreement with previous findings^[91]34–[92]36, we observed that the
   BioCyTASQ biomimetic probe, previously shown to highlight cytoplasmic
   RG4s foci^[93]21, colocalized with the OMM protein TOMM20 (Fig. [94]1a,
   b) or the mitochondrial marker (MitoTracker Deep Red) in HeLa cells
   (Supplementary Fig. [95]1c) but was lost after RNAse treatment
   (Supplementary Fig. [96]1d). This result together with the observation
   that TOMM20 did not colocalize with a non-mitochondrial cytosolic
   protein (Supplementary Fig. [97]1e, f) suggests that RG4s probes mapped
   RG4s at mitochondria. As expected, the BioCyTASQ signal increased after
   addition of the G4-stabilizing ligand PhenDC3 (Fig. [98]1a, b,
   Supplementary Fig. [99]1c, g). However, this was accompanied by a
   change in colocalization with mitochondria due to the spreading of the
   RG4 signal toward the cytosolic area (Fig. [100]1a, b, Supplementary
   Fig. [101]1c). Overall, these data suggest that some cytosolic RG4s
   mapped to mitochondria and their stabilization increased RG4 formation,
   which is accompanied by a modification in their proportional
   co-distribution. Next, we asked if PhenDC3 impacted mitochondrial
   functions and biogenesis in three cancer cell lines (HeLa, HCT116 and
   SKMEL28). In HCT116 cells, we found that PhenDC3 reduced the oxygen
   consumption rate (OCR) as measured by Seahorse assays (Fig. [102]1c)
   and the membrane potential (Fig. [103]1d), without affecting cell
   viability and number after 24 h treatment (Supplementary Fig. [104]2a).
   In agreement with previous findings^[105]35, PhenDC3 did not modify
   mitochondrial mass (Supplementary Fig. [106]2b). Consistent with the
   emerging notion that proliferation depends on mitochondrial
   activity^[107]42,[108]43, we observed that PhenDC3 inhibited
   proliferation of the three cell lines from day 4 with effects whose
   magnitude is correlated with mitochondrial respiratory function
   (Fig. [109]1c, e, Supplementary Fig. [110]2c, d). Together, these
   results indicate that RG4s are important cis-elements regulating the
   cytoplasmic expression of nuclear-encoded mitochondrial mRNAs and that
   modulation of their conformation affects both mitochondrial activity
   and cancer cell proliferation.

Fig. 1. RG4 localization and function in mitochondria.

   [111]Fig. 1
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   a RG4 detection in fixed cells using the BioCyTASQ probe (1 µM for
   24 h) coupled with TOMM20 immunofluorescence in HeLa cells treated with
   DMSO or 20 µM of PhenDC3 for 24 h. Scale bar: 10 μm. Shown is a
   representative result from n = 3 independent experiments. b Graphs
   displaying the fluorescence intensity (arbitrary unit) in each channel
   over the distance depicted by the white arrow in (a). The correlation
   coefficient (R^2) of the colocalized fluorescence intensities of
   BioCyTASQ with TOMM20 signal is plotted. Data are presented as mean
   values ± SEM for 5 or 6 different cells, P-value = 0.0024. c
   Quantification of the normalized Oxygen Consumption Rate (OCR) per
   cell, measured by a Seahorse assay, in HCT116 cells treated with DMSO
   or a dose scale of PhenDC3 for 16 h. Data are presented as mean
   values ± SEM of n = 3 independent experiments. d Quantification of the
   Mitochondrial Membrane Potential (MMP) using flow cytometry analysis of
   the fluorescent TMRE probe in HCT116 cells treated with DMSO or PhenDC3
   for 16 h or 48 h and plotted relatively to the DMSO condition. Data are
   presented as mean values ± SEM of n = 3 independent experiments,
   P-value = 0.0135 and P-value = 0.0373 for 16 h and 48 h PhenDC3
   treatment, respectively. e Quantification of the Oxygen Consumption
   Rate (OCR) linked to basal and maximal mitochondrial respiration, ATP
   turnover, proton leak, spare respiratory capacity, measured by a
   Seahorse assay, and of the proliferation in HCT116, HeLa and SKMEL28
   cells treated with DMSO or a dose scale of PhenDC3 for 16 h. Data are
   presented as mean values ± SEM of n = 2 (for proliferation assay in
   HeLa) or n = 3 independent experiments. For all the panels, *P < 0.05,
   **P < 0.01, ***P < 0.001, ns: non-significant (two-sided paired t-test
   for OCR and two-way ANOVA for the proliferation). For (b, c, d, e)
   Source data are provided as a [113]Source Data file indicating exact
   P-values for (e).

RG4s are molecular determinants of OMM-localized translation

   Since nuclear-encoded mitochondrial mRNAs could partly localize and
   undergo translational regulation at the outer membrane of mitochondria
   (OMM)^[114]15,[115]44,[116]45, we analyzed whether RG4-containing mRNAs
   exhibit a specific spatial distribution using data from APEX-seq, a
   method based on direct proximity labeling of RNA using the peroxidase
   enzyme APEX2^[117]7. We found that RG4-containing mRNAs were more
   enriched at the OMM compared to the ERM (Endoplasmic Reticulum
   Membrane) (Supplementary Fig. [118]3a). Using RNA immunoprecipitation
   (RIP) assays with HCT116 cytoplasmic extracts and the BG4
   antibody^[119]20, we validated that a subset of these OMM-associated
   mRNAs (for which RG4s were identified in cellulo^[120]18 and/or in
   vitro^[121]19) was prone to form RG4s in cellulo (Fig. [122]2a,
   Supplementary Fig. [123]3b). We found that PhenDC3 modified the protein
   expression but not the levels of these RG4-containing mRNAs
   (Fig. [124]2b, c and Supplementary Fig. [125]3c), suggesting that RG4
   stabilization affects the translation of OMM-associated mRNAs. Finally,
   given that mitochondrial translation depends on the coordination
   between the cytoplasmic and the mitochondrial machinery^[126]46, we
   tested whether the deregulation of RG4-dependent cytoplasmic
   translation could have an impact on the expression of proteins encoded
   by the mitochondrial genome via RG4-less mRNAs and translated by
   mitoribosomes. We found that PhenDC3 preferentially decreased COX-1 as
   compared to COX-4 (Supplementary Fig. [127]3d), which are
   mitochondria-encoded and nuclear-encoded subunits of the complex IV in
   the electron transport chain in mitochondria, respectively.

Fig. 2. RG4s are molecular determinants of OMM-localized translation.

   [128]Fig. 2
   [129]Open in a new tab

   a Immunoprecipitation (IP) of in cellulo RNA-protein complexes using
   the BG4 antibody or control IgG, followed by RT–qPCR analysis. Fold
   changes are indicated. Data represent n = 2 or n = 3 independent
   experiments in duplicate when 4 or 6 dots are plotted, respectively. b
   Western blot analysis in HCT116 cells treated with PhenDC3 for 48 h. c
   Fold enrichment of protein/mRNA levels from (b). d AKAP1 mRNA/protein
   localization analysed by RNAscope and immunofluorescence (IF). (e)
   Quantification from (d). P-value = 0.0297 or 0.0425 for PhenDC3 or Puro
   treatment, respectively (two-sided paired t-test). f RNAscope analysis
   of AKAP1 and MTND5 mRNA localization as in (d). g Quantification from
   (f). h Proximity Ligation Assay coupled with puromycin Labeling
   (PLA-Puro) of AKAP1 or IkB, combined with TOMM20 IF. Graphs displaying
   the fluorescence intensity in each channel over the distance are
   depicted. i Quantification from (h). P-value = 0.0125 for puro-AKAP1
   after PhenDC3 treatment. j Ratio of Renilla/Firefly luciferase
   activities (Rluc/Fluc) after transfection of RLuc-GALNT2 mRNA reporters
   (depicted in Supplementary Fig. [130]3j) containing the RG4 unmodified
   (WT), mutated (Mut) or 7-deaza-modified (7dG) for n = 4 independent
   experiments. k Quantification of the colocalization between TOMM20 and
   PLA-Puro dots from in vitro transcribed Rluc-GALNT2 reporter mRNA
   containing the RG4 WT, Mut or 7dG from images in Supplementary
   Fig. [131]2i. P-value = 0.0243 for the Puro-Rluc. In (a, c, j), data
   represent mean ± SEM. In (e, g, i, k), boxes extend from 25th to 75th
   percentiles; middle line is the median, and “+” is the mean; the
   whiskers show upper and lower extremes. For (a, b, c, e, g, i, j, k)
   Source Data are provided as a [132]Source Data file indicating exact
   P-values for (a, c, j). In (b, c), the blot shown is representative of
   n = 3 independent experiments. In (d, f, h) a representative field from
   n = 3 independent experiments is shown. In (e, g, i, k), data represent
   n = 3 independent experiments and each dot is a cell. In (a, c, g, j,
   k), *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns
   non-significant (two-sided paired t-test).

   The prevailing consensus on nuclear-encoded mitochondrial mRNA
   expression is that their translation, often relying on 3’UTR
   sequences^[133]47, is accomplished by OMM-associated ribosomes^[134]48.
   Their recruitment at this specific location may depend on the assembly
   of the translation machinery (ribosome-dependent mechanism) or rely on
   specialized RBPs responsible for localizing them in the vicinity of the
   mitochondria (ribosome-independent mechanism)^[135]7. Nuclear-encoded
   RG4-containing mitochondrial mRNAs showed higher enrichment in the
   ribosome-dependent group (Supplementary Fig. [136]3e), with a
   predominant location of the RG4s in the 3’UTR (Supplementary
   Fig. [137]3f). To define whether RG4s are molecular determinants of
   OMM-localized translation, we started by monitoring an endogenous
   ribosome-dependent mRNA. We focused on AKAP1, a scaffolding protein
   that acts as a mitochondrial signaling hub by recruiting protein kinase
   A (PKA) to the OMM and regulates mitochondrial biogenesis, oxidative
   metabolism, and cell survival^[138]49. Indeed, AKAP1 mRNA was found to
   colocalize with its encoded protein at mitochondria in a
   translation-dependent manner, suggesting that this mRNA could be
   translated locally^[139]15. Specifically, AKAP1 mRNA was delocalized
   and lost its colocalization with the encoded protein after treatment
   with puromycin, a translation inhibitor^[140]15. As we observed that
   PhenDC3 induced an uncoupling between cellulo RG4s and mitochondria
   (Fig. [141]1a, b, Supplementary Fig. [142]1c), we asked whether the
   ligand-induced stabilization of the AKAP1 RG4s modified the
   translation-dependent localization of this mRNA to mitochondria. To
   this end, we used RNAscope^[143]50 for the detection of AKAP1 mRNA and
   the MTND5 mitochondrial mRNA combined with immunofluorescence
   visualization of AKAP1 protein. As shown in Fig. [144]2d, e, puromycin
   induced a 20% loss of colocalization between the AKAP1 mRNA and the
   encoded protein comparable to that observed previously^[145]15, with
   similar effects induced by PhenDC3 treatment. In agreement with
   previous findings^[146]7 and with the results in Fig. [147]1a, b and
   Supplementary Fig. [148]1c that show ligand-induced uncoupling between
   the RG4 signal and mitochondria marker, we found that the association
   of AKAP1 mRNA with mitochondria was reduced in the presence of PhenDC3
   to the same extent as in the presence of the translation inhibitor
   puromycin (Fig. [149]2f, g). Consistent with this, analysis of the
   distribution of AKAP1 mRNA colocalizing with the encoded protein
   between the mitochondria (MTND5 mRNA) and the cytosol revealed a
   predominant association with the mitochondria and a decrease in this
   fraction after treatment with the ligand, accompanied by a proportional
   increase in AKAP1 RNA/protein colocalization in the cytosol
   (Supplementary Fig. [150]3g). Similar losses of colocalization were
   observed for two other nuclear-encoded mitochondrial transcripts
   (Supplementary Fig. [151]3h) that contain RG4s (Fig. [152]2a). To
   strengthen the conclusion that PhenDC3 inhibits OMM-localized AKAP1
   mRNA translation, we combined immunofluorescence visualization of
   TOMM20 with proximity ligation assay (PLA) detection of neo-synthesized
   AKAP1. We used antibodies against N-term AKAP1 and puromycin which is
   covalently incorporated into nascent protein chains. Consistent with
   Fig. [153]2d–g, we found that the PLA signal for the neo-synthetized
   AKAP1 was localized at the OMM, as opposed to the non-mitochondrial
   protein IkB (Fig. [154]2h, i and Supplementary Fig. [155]3i). PhenDC3
   reduced both the number of PLA dots for the nascent AKAP1
   (Supplementary Fig. [156]3j) and their OMM localization (Fig. [157]2i).
   Overall, these results indicate that RG4 stabilization decreased AKAP1
   OMM-localized neo-synthesis.

   To provide more direct evidence of RG4 involvement in OMM-localized
   translation, we generated mitochondria localization-specific mRNA
   reporters containing the 3’UTR of GALNT2, a nuclear-encoded
   mitochondria mRNA in which (experimentally identified^[158]18,[159]19)
   RG4s were either wild-type or mutated (Supplementary Fig. [160]3k). We
   observed that RG4 mutations reduced the luciferase expression in both
   Hela and HCT116 cells (Fig. [161]2j). Similar results were obtained
   with a reporter that did not form RG4s due to the guanines being
   replaced by the analogue 7-deaza-guanines (Fig. [162]2j), which
   prevents Hoogsteen base pairing and RG4 formation^[163]51. In agreement
   with this, colocalization between TOMM20 and PLA detection of
   neo-synthesized luciferase reporters revealed a reduction in
   mitochondrially localized translation when the RG4 was unable to fold,
   being either mutated or containing the guanine analogue (Fig. [164]2k
   and Supplementary Fig. [165]3l). In line with this, RG4 formation
   decreased the colocalization between the GALNT2 mRNA reporter and the
   MTND5 mitochondrial mRNA (as revealed by RNAscope analysis)
   (Supplementary Fig. [166]3m). Taken together, these results suggest
   that RG4s are important determinants of OMM-localized translation and
   that the impairment of their dynamics between the folded and unfolded
   states interferes with this function, with consequences for
   mitochondrial respiration. Similarly to PhenDC3, treatment with cPDS
   induced RG4 stabilization (Supplementary Fig. [167]4a) and resulted in
   both reduced mitochondrial respiration (Supplementary Fig. [168]4b, c)
   and association of GALNT2 with the mitochondria (Supplementary
   Fig. [169]4d), indicating that the role of RG4 dynamics in the
   crosstalk between protein synthesis and energy metabolism is
   ligand-independent.

hnRNP U binds to RG4-containing nuclear-encoded mitochondrial mRNAs

   To gain insights into the proteins that could bind these RG4s, we
   reasoned that this factor must be both an RNA- and RG4-binding protein
   associated with the translation machinery. We thus intersected the list
   of RBPs interacting with the 3’UTRs of RG4-containing
   ribosome-dependent nuclear-encoded mitochondrial mRNAs derived by
   ENCODE eCLIP (enhanced crosslinking immunoprecipitation)-data
   sets^[170]52, the catalog of RG4-binding proteins^[171]39 and the
   ribosome-associated proteins^[172]53. This analysis revealed that the
   hnRNP U, RPS3, and YBX3 RBPs could be strong candidates for binding to
   RG4s of nuclear-encoded mitochondrial mRNAs in a ribosome-dependent
   manner (Supplementary Fig. [173]5a). To further explore the regulatory
   mechanism of RG4s at OMM, we thus focused on hnRNP U as it was
   previously shown to play a role in energy metabolism^[174]54,[175]55.

   hnRNP U is a protein of the hnRNP family, mainly described as a
   regulator of nuclear post-transcriptional steps of mRNA
   maturation^[176]56–[177]59. However, its function related to RG4
   regulation and mRNA translation has not been investigated yet. In
   agreement with previous data reporting the function of hnRNP U in the
   cytoplasm^[178]60–[179]64, we observed that, beyond its presence in
   nuclear fractions, hnRNP U was enriched in cytosolic and microsomal
   fractions (Fig. [180]3a). To investigate its binding to cytoplasmic
   RG4-containing mRNAs, we first reanalyzed previously published in
   cellulo hnRNP U-RNA interactions using crosslinking and
   immunoprecipitation (CLIP) of hnRNP U from cytoplasmic fractions
   followed by deep RNA sequencing (cyto-CLIP^[181]62). Strikingly, 11.7%
   of 793 hnRNP U-bound mRNAs in the cytoplasm were associated with
   mitochondria (Fig. [182]3b) and significantly associated with metabolic
   reprogramming in cancer by pathway enrichment analysis (Fig. [183]3c).
   Consistently, hnRNP U immunoprecipitation with cytoplasmic extracts
   followed by RT-qPCR analysis revealed that hnRNP U associated with
   nuclear-encoded mitochondrial mRNAs in cellulo (Fig. [184]3d,
   Supplementary Fig. [185]5b). To determine if hnRNP U binds to RG4s, we
   first used RNA affinity chromatography with cytoplasmic extracts.
   Consistent with a recent proteomic screen for cytoplasmic proteins
   binding to folded/unfolded RG4s^[186]26, we found that hnRNP U
   preferentially bound to canonical (G3A2G3A2G3A2G3) or naturally
   occurring sequences (i.e., the RG4-forming sequence in the 5’UTR of
   NRAS^[187]25) over RNAs that do not form RG4s due to the replacement of
   guanines with 7-deaza-guanines (Fig. [188]3e and Supplementary
   Fig. [189]5c). Furthermore, previous RNA affinity purification followed
   by mass spectrometry (RP-MS) experiments identified hnRNP U as being
   preferentially associated with wild-type as compared to sequences
   lacking G-runs^[190]27–[191]29,[192]31 or forming hairpin
   structures^[193]24. These results thus support the conclusion that
   hnRNP U is an RG4-binding protein preferentially binding to folded
   RG4s. To explore these results further, we intersected the hnRNP U
   cyto-CLIP dataset with both in vitro validated RG4s (datasets from Guo
   et al.^[194]18 and Kwok et al.^[195]19) and in silico predicted
   RG4s^[196]39. As shown in Fig. [197]3f, the hnRNP U-bound 3’UTR and CDS
   regions contain a similar proportion of experimental (based on Kwok et
   al.^[198]19) and predicted RG4s, while the proportion in the 5’UTR was
   consistently smaller compared to CDS/3’UTR. We found that 44.64% and
   34.68% of hnRNP U-bound mRNAs contained experimentally validated RG4s
   from the Kwok^[199]19 and Guo datasets^[200]18, respectively, as
   extracted from the QUADRatlas database^[201]39. This proportion
   increases to 61% when considering only the high-scoring RG4 forming
   sequences predicted with QGRSmapper^[202]65 (score ≥ 19). Importantly,
   hnRNP U-bound mRNAs containing RG4s identified experimentally or in
   silico predicted were significantly associated with metabolic
   reprogramming pathways (adjusted P-value 3.2E-08 (Kwok dataset),
   2.5E-07 (Guo dataset), 9.4E-06 (predicted)). In addition, analysis of
   the density of hnRNP U binding sites around RG4s (based on Kwok et
   al.^[203]19) indicated significant colocalization for the CDSs and the
   3’UTRs but not for the 5’UTRs (Fig. [204]3g). To define whether this
   pattern is specific to hnRNP U, we looked at the density of binding
   around RG4s for other ENCODE eCLIP-profiled RBPs. We found that 62/132
   RBPs (47%) do not have the same binding pattern as hnRNP U (as
   exemplified by the RBP NCBP2 (Supplementary Fig. [205]5d)). Finally,
   RIP assays with cytoplasmic extracts and the BG4 antibody confirmed
   that several nuclear-encoded mitochondrial mRNAs targeted by hnRNP U
   can form RG4s in cellulo (Fig. [206]3h). Based on the “bind, unfold and
   lock” model^[207]26, whereby RG4-BPs synergize with helicases (“bind”)
   to unwind RG4s (“unfold”) and promote G-rich-BP binding that keeps RG4s
   unfolded (“lock”), the hnRNP U-RG4s interaction could cooperate with
   RNA helicases, thus inducing RG4 melting. In support of this
   hypothesis, we found that RG4 foci visualized using the BioCyTASQ
   increase after hnRNP U silencing (Fig. [208]3i, j). This is consistent
   with what we observed after treatment with PhenDC3 (Fig. [209]1a, b,
   Supplementary Fig. [210]1c, g). Overall, these results suggest that
   cytoplasmic hnRNP U binds nuclear-encoded RG4-containing mitochondrial
   mRNAs with consequences on the RG4 structural equilibrium.

Fig. 3. hnRNP U binds nuclear-encoded RG4-containing nuclear-encoded
mitochondrial mRNAs.

   [211]Fig. 3
   [212]Open in a new tab

   a Subcellular fractionation of HCT116 cells, followed by western blot
   analysis. Lamin A: nuclear marker, Tubulin: cytoplasmic marker
   associated with microsomes. Nuclear (N), perinuclear (PN), microsomal
   (M), and cytosolic fractions (C). Shown is a representative result from
   n = 3 independent experiments. b Proportion of hnRNP U binding sites in
   5’UTR, CDS, and 3’UTR and percentage of mitochondrial mRNAs (mito)
   bound by hnRNP U in each region. c −log10(P-value) of the enrichment
   analysis of hnRNP U targets^[213]61. P-value computed from Fisher exact
   test. d Immunoprecipitation of cellulo RNA-protein complexes (RIP)
   using the hnRNP U antibody, followed by RT-qPCR analysis. Fold changes
   are indicated in italics. e RNA affinity chromatography using the G3A2
   and NRAS RG4s native (WT) or 7-deaza-modified (7dG), followed by
   western blot analysis. Shown is a representative result from n = 6 and
   n = 2 independent experiments for the G3A2 and NRAS RG4s, respectively.
   f Proportion of hnRNP U binding sites in 5’UTR, CDS, and 3’UTR
   containing experimental and predicted RG4s. g Density of hnRNP U
   binding sites around RG4s located in mRNAs (all mRNA), 5’UTRs, CDSs,
   and 3’UTRs. At distance 0 and until +30 −80: P = 0.004 for CDS, P = 0
   for 3’UTR, P = 1 for 5’UTR (Bootstrap test with 1000 sets of random
   sites). h RIP using the BG4 antibody, followed by RT–qPCR analysis.
   Fold changes are indicated in italic. USP1: positive control^[214]26. i
   RG4 detection after control (Ctr) or hnRNP U siRNAs. Scale bar: 20 μm.
   Shown is a representative result from n = 4 independent experiments. j
   Quantification of signal intensities from (i), each dot is a cell,
   P-value = 0.000181 (two-sided paired t-test). For (a, d, e, h, j)
   source data are provided as a [215]Source Data file indicating exact
   P-values for (d) and (h). When indicated, *P < 0.05, **P < 0.01,
   ***P < 0.001, ns: non-significant (two-sided paired t-test). In (d, h),
   data are presented as mean values ± SEM of n = 2 or n = 3 independent
   experiments in duplicate when 4 dots or 6 dots are plotted,
   respectively.

hnRNP U localizes to the OMM and interacts with the translational machinery
and nuclear-encoded mitochondrial proteins

   To further explore the role of hnRNP U when bound to RG4-containing
   nuclear-encoded mitochondrial mRNAs, we reasoned that it should be
   localized in OMM compartments and possibly interact with the factors
   therein contained, i.e. nuclear-encoded mitochondrial mRNAs to be
   translated, ribosomes, translational regulators, and mitochondrial
   proteins to be imported into mitochondria^[216]66,[217]67. To address
   these possibilities, we purified mitochondria and determined whether
   hnRNP U was associated with these organelles. We observed that hnRNP U
   was found in the cytoplasm, microsomes, and mitochondrial fractions
   (Fig. [218]4a) like other proteins associated with OMM (TOMM20),
   residing in the mitochondrial matrix (DHX30, GRSF1), or associated with
   the inner mitochondrial membrane (OXPHOS), but unlike other RG4-BPs
   (DDX5) or known hnRNP U interactors (PRMT1^[219]68). Then, we asked
   whether hnRNP U could be associated with the OMM using the protease
   protection assay after mitochondria purification. In the absence of a
   detergent that solubilizes the mitochondrial membrane (Triton-X), the
   addition of proteinase K removed hnRNP U from the mitochondrial
   fraction, suggesting that hnRNP U is an OMM-interacting protein
   (Fig. [220]4b). DDX3X, a RG4 helicase known to play a role in mRNA
   translation and mitochondrial respiration^[221]69, showed a proteinase
   K protection profile similar to that of hnRNP U (Fig. [222]4b). Unlike
   hnRNP U and DDX3X, a fraction of cytoplasmic GRSF1 and DHX30, two RBPs
   involved in mRNA metabolism within
   mitochondria^[223]38,[224]70,[225]71, was partially resistant to
   proteases but completely degraded when mitochondria were permeabilized
   with Triton-X (Fig. [226]4b). These results suggest that hnRNP U and
   DDX3X do not reside in mitochondria but rather are associated with the
   OMM where they can interact with RG4-containing mRNAs. This is in
   agreement with a proteomic analysis of purified mitochondria from
   HCT116^[227]67. Indeed, consistent with Fig. [228]3e and our previous
   proteomic screen for proteins binding folded/unfolded RG4s^[229]26, RNA
   pull-down performed with mitochondrial (Supplementary Fig. [230]6a) or
   OMM fractions (Fig. [231]4c) showed that hnRNP U and DDX3X interact
   preferentially with folded RG4s in these fractions. In contrast, GRSF1,
   which promotes RG4 melting^[232]38 and binds to G-rich
   sequences^[233]72, showed a stronger association with G-rich sequences
   unable to form RG4s (Fig. [234]4c, Supplementary Fig. [235]6a).

Fig. 4. hnRNP U protein interactors and sub-cellular localization.

   [236]Fig. 4
   [237]Open in a new tab

   a Subcellular fractionation of HCT116 cells to obtain Total (T),
   Microsomal (M), and Mitochondrial (Mi) fractions, followed by western
   blot analysis. Shown is a representative result from n = 8 independent
   experiments for hnRNP U and DDX3X, n = 5 independent experiments for
   RPS6, OXPHOS complex and GRSF1, n = 6 independent experiments for
   TOMM20 and DHX30, n = 2 independent experiments for PRMT1 and DDX5. b
   Protease protection assay after mitochondria purification of HCT116
   cells followed by western blot analysis of the indicated proteins. TX:
   Triton-X. PK: Proteinase K. Shown is a representative result from n = 2
   independent experiments. c RNA affinity chromatography using the G3A2
   sequences either native (WT) or 7-deaza-modified (7dG) and HCT116 OMM
   (Outer Mitochondrial Membrane) extracts, followed by western blot
   analysis. Shown is a representative result from n = 4 independent
   experiments for hnRNP U and GRSF1 and n = 3 independent experiments for
   DDX3X. d Distribution of hnRNP U partners identified by
   immunoprecipitation (IP) cytoplasmic HCT116 cell extracts, followed by
   mass spectrometry (IP-MS, n = 4 independent experiments). P = 1.58e-46,
   P = 5.01e-46, and P = 1.58e-22 for the mitochondrial proteins, the
   proteins involved in the cytoplasmic translation, and the RG4-BPs,
   respectively (Fisher test). For (a, b, c), source data are provided as
   a [238]Source Data file.

   Then, we analyzed the hnRNP U protein interactome by performing
   immunoprecipitation of hnRNP U partners from cytoplasmic fractions in
   the presence of benzonase, an endonuclease that removes nucleic acids.
   Proteins co-immunoprecipitated with hnRNP U were subjected to tryptic
   digestion followed by higher-energy collisional dissociation-tandem
   mass spectrometry (HCD-MS/MS), thus allowing quantitative label-free
   proteomic analysis of protein–protein interaction data^[239]73. The
   results were evaluated using significance analysis of interactome
   (SAINT) analysis^[240]74, a computational tool that assigns confidence
   scores to protein–protein interactions, with hnRNP U interactors
   defined as proteins with SAINT score SP ≥ 0.8. This quantitative
   analysis with four replicates enabled the identification of 134
   proteins that interact with hnRNP U in an RNA-independent manner
   (Fig. [241]4d, Supplementary Data [242]1), assigned to specific
   functional pathways such as translation and mitochondrial translation
   (Supplementary Fig. [243]6b). Among these proteins (Fig. [244]4d), we
   found 1) ribosomal proteins of the cytoplasmic and mitochondrial
   translational machineries, some of which were validated by
   immunodetection (Supplementary Fig. [245]6c), and 2) 26 RG4-binding
   proteins, including DDX3X, which, similarly to hnRNP U, was associated
   with the OMM (Fig. [246]4b) and bound RG4s at this specific subcellular
   location (Fig. [247]4c, Supplementary Fig. [248]6a). Taken together,
   these results suggest that a fraction of hnRNP U is localized at the
   OMM where it interacts with both the translational machinery (including
   ribosomes, RG4 binding proteins, and helicases as DDX3X) and
   nuclear-encoded mitochondrial proteins.

hnRNP U regulates the OMM-localized protein synthesis of RG4-containing mRNA
targets

   The observation that hnRNP U was found in the cytoplasm (Fig. [249]3a)
   where it interacts with ribosomal proteins and translational regulators
   (Fig. [250]4d) prompted us to study its role in translation. To this
   end, we performed polysome profiling combined with immunoblotting to
   monitor the distribution of hnRNP U in translationally inactive and
   active fractions in the presence or absence of puromycin, a
   drug-inducing ribosome dissociation. We found that hnRNP U
   co-sedimented with translating polyribosomes and that their association
   depended on polysome integrity (Fig. [251]5a). To demonstrate a
   functional role for hnRNP U in the regulation of translation, we
   quantified the global protein synthesis after hnRNP U siRNA-mediated
   silencing by pulsed puromycin labeling and immunoblotting with an
   anti-puromycin antibody (i.e. SUnSET). We found that the depletion of
   hnRNP U did not modify the overall translation rates (Supplementary
   Fig. [252]7a, b) while the polysomal profile was only slightly altered
   by hnRNP U depletion (Supplementary Fig. [253]7c, d), indicating that
   hnRNP U-deficient cells are not globally deficient in protein
   synthesis. Since in these conditions cellular proliferation was not
   affected (Supplementary Fig. [254]7e), changes in translational
   efficiency did not depend on this process. Based on these results and
   our previous findings showing hnRNP U binding to RG4-containing
   nuclear-encoded mRNAs (Fig. [255]3d, f, g, h), we tested whether
   depletion of hnRNP U could selectively control the translation of these
   mRNAs. To this end, we performed polysomal fractionation of hnRNP
   U-depleted cells, followed by RNA isolation from non-polysome (NP),
   light (LP) and heavy (HP) polysome fractions, and then by either RNA
   sequencing (Supplementary Fig. [256]7f) and/or RT-qPCR analysis
   (Fig. [257]5b, Supplementary Fig. [258]7g, h). Following hnRNP U
   silencing, the translational efficiency of these mRNAs was quantified
   either by analyzing the HP/total mRNA ratio (Fig. [259]5b,
   Supplementary Fig. [260]7g) or by measuring the distribution of each
   mRNA across the gradient (Supplementary Fig. [261]7h). Focusing first
   on previously characterized RG4-containing nuclear-encoded
   mitochondrial mRNAs bound by hnRNP U (Fig. [262]3d), we observed that
   hnRNP U depletion induced a significant decrease in mRNA association
   with translating polysomes (Fig. [263]5b, Supplementary Fig. [264]7g,
   h), indicating a role of hnRNP U in translational activation of a
   subset of nuclear-encoded mitochondrial mRNAs. To strengthen these
   results and provide evidence of direct involvement of hnRNP U in mRNA
   translation, we performed in vitro translation using purified
   recombinant hnRNP U and a luciferase reporter transcript containing the
   3’UTR of GALNT2 (Supplementary Fig. [265]3g), an RG4-containing
   transcript (Fig. [266]2a) bound (Fig. [267]3d) and translationally
   regulated by hnRNP U (Fig. [268]5b, Supplementary Fig. [269]7h). We
   found that hnRNP U addition increased the translation of the mRNA
   reporter containing the wild-type RG4 but not the mutated version, nor
   the luciferase control transcript (Fig. [270]5c). Furthermore, similar
   to PhenDC3 treatment (Fig. [271]2d, e), hnRNP U silencing reduced the
   colocalization between AKAP1 mRNA, which is a translational target of
   hnRNP U (Fig. [272]5b, Supplementary Fig. [273]7h), and the encoded
   protein (Fig. [274]5d–g), suggesting that hnRNP U regulates
   OMM-localized mRNA translation. To study the extent of translational
   regulation of nuclear-encoded mitochondrial mRNAs by hnRNP U, we
   analyzed the translational targets of hnRNP U at the transcriptome-wide
   level using polysomal profiling followed by RNA sequencing. We
   identified 60 RG4-containing nuclear-encoded mitochondrial transcripts
   whose association with HP fractions was altered after hnRNP U
   depletion, a subset of which was validated by RT-qPCR (Supplementary
   Fig. [275]7f, g, h). Next, we defined whether the hnRNP U-dependent
   mechanism of mitochondrial mRNA translational regulation could be tuned
   in response to stimuli inducing mitochondrial metabolic switches. A
   strong candidate for this function is the mechanistic/mammalian
   serine/threonine kinase target of rapamycin (mTOR) as its inhibition
   selectively inhibited the translation of a subset of nuclear-encoded
   mitochondrial mRNAs^[276]75 and reduced the association of hnRNP U with
   mRNAs^[277]76. In agreement with previous results on stress-induced RG4
   folding in cancer cells^[278]77, we observed that rapamycin, an mTOR
   inhibitor mimicking the effect of starvation, induced a significant
   increase in RG4 structuration (Supplementary Fig.8a). Importantly,
   rapamycin or Torin 1, a very potent and selective mTOR inhibitor,
   reduced the colocalization between AKAP1 mRNA and the encoded protein
   (Fig. [279]5f, g and Supplementary Fig. [280]8b). Finally, we found
   that hnRNP U binding to RG4s (Fig. [281]5h, i) and the translation
   machinery (Supplementary Fig. [282]8c) was inhibited by Torin 1,
   possibly through altered phosphorylation of hnRNP U (Supplementary
   Fig. [283]8d, e). Together, these results suggest that hnRNP U is
   associated with the translational machinery and regulates the
   OMM-localized protein synthesis of RG4-containing mRNA targets with
   functions in mitochondria. Importantly, our data suggest the existence
   of a mTOR-hnRNP U-mitochondrial translation axis that modulates
   cellular energy production by mitochondria. The underlying mechanism
   involves mTOR stimulating the selective translation of nuclear-encoded
   mitochondrial mRNAs by modulating RG4 formation as well as hnRNP U
   phosphorylation and polysomal association and its interaction with
   RG4-containing mRNAs.

Fig. 5. hnRNP U is involved in translation regulation of RG4-containing
nuclear-encoded mitochondrial mRNAs.

   [284]Fig. 5
   [285]Open in a new tab

   a Polysome profile of HCT116 cells DMSO or puromycin (Puro) treated,
   followed by western blot analysis. RPS6: positive control. Shown is a
   representative result from n = 2 independent experiments. b RT-qPCR
   analysis from non-polysomal (NP), light (LP) and heavy (HP) polysomal
   fractions from Supplementary Fig. [286]7c. *P < 0.05, **P < 0.01,
   ***P < 0.001, ns: non-significant (two-sided paired t-test). c In vitro
   translation of Rluc-GALNT2 reporter mRNAs (depicted in Supplementary
   Fig. [287]3j) containing the RG4 unmodified (WT), or mutated (Mut) and
   of the control Rluc reporter mRNA with or without recombinant hnRNP U.
   d RNAscope analysis of AKAP1 mRNA localization and immunofluorescence
   (IF) analysis of AKAP1 protein localization in HeLa cells treated with
   control (Ctr) or hnRNP U siRNAs. Scale bar: 20 μm. e Quantification
   from images in (d). Data represent n = 3 independent experiments and
   each dot is a cell, P-value = 0.02486 (two-sided paired t-test). (f)
   RNAscope and IF analysis as in (d) in HeLa cells treated 20 µM PhenDC3
   for 24 h or 300 nM Torin 1 for 2 h. Scale bar: 10 μm. g Quantification
   from images in (f). Data represent n = 3 and n = 2 independent
   experiments for the Puro, Torin 1 and PhenDC3 treatments, respectively,
   each dot is a cell. h RNA affinity chromatography using RG4 sequences
   either native (WT) or 7-deaza-modified (7dG) and cytoplasmic extracts
   of HCT116 treated with DMSO or 300 nM Torin 1 for 3 h, followed by
   western blot analysis. i Quantification of the protein levels on the
   RG4 WT from (h). P-value = 0.02336, ns non-significant (two-sided
   paired t-test). For (a, b, c, e, g, h), Source Data is provided as a
   [288]Source Data file indicating exact P-values for (b, c, g). Data are
   presented as mean values ± SEM of n = 3 for (b), (c), n = 4 for (i)
   independent experiments. For (e, g), the box extends from 25th to 75th
   percentiles; middle line is the median and “+” is the mean; the
   whiskers show upper and lower extremes. For (d, f), shown is a single
   representative field over n = 3 independent experiments.

hnRNP U affects oxidative metabolism by regulating the synthesis of OXPHOS
complex proteins

   OXPHOS, the main energy source of the cell, involves five multisubunit
   protein complexes harboring more than 80 nuclear-encoded proteins, as
   well as 13 mitochondrial-encoded subunits (complexes I, III, IV, and
   V). According to a recently proposed model of mitochondrial
   translational plasticity^[289]78, the synthesis of the latter adapts to
   the influx of nuclear DNA-encoded OXPHOS subunits. Based on this model
   and taking into account that hnRNP U interacts with mRNAs and proteins
   (Fig. [290]3b, d) whose products are imported into the mitochondria, we
   asked whether hnRNP U shaped OXPHOS assemblies and affected
   mitochondrial functions. We observed that hnRNP U silencing reduced the
   expression of proteins belonging to the five OXPHOS complexes,
   including both nuclear-encoded (ATP5A, UQCRC2, SDHB, NDUFB8) and
   mitochondria-encoded proteins (COX2) (Fig. [291]6a, b). We then
   determined whether hnRNP U-induced altered expression of OXPHOS
   complexes resulted in modified OXPHOS enzymatic activity measured by
   OCR rates using Seahorse assays. Figure [292]6c, d shows that hnRNP U
   depletion significantly impairs mitochondrial respiration with reduced
   basal OCR and lowered ATP turnover rate. We also tested the effect of
   hnRNP U depletion on glycolysis because (1) the inhibition of OXPHOS
   function might induce a metabolic shift to glycolysis-mediated energy
   production, also called Warburg effect^[293]79, and (2) hnRNP U-bound
   mRNAs are significantly associated with glycolysis-related pathways
   (Fig. [294]3c). Using the Seahorse mitochondrial and glycolytic stress
   tests as a measure of glycolysis, we found that silencing of hnRNP U
   inhibited glycolysis (Fig. [295]6e, Supplementary Fig. [296]9a). Since
   DDX3X, similarly to hnRNP U, was found at the OMM (Fig. [297]4a, b),
   interacts with RG4s (Fig. [298]4c, Supplementary Fig. [299]6a) and was
   shown to modulate mitochondrial respiration^[300]69, we tested the
   effect of silencing DDX3X alone or in combination with hnRNP U on both
   mitochondrial respiration and glycolysis. We observed that DDX3X and
   hnRNP U similarly reduced mitochondrial respiration and glycolysis, and
   that combined depletion led to a further reduction, though not in a
   synergistic manner (Fig. [301]6c, d, Supplementary Fig. [302]9a). We
   also found that the effect of hnRNP U silencing on mitochondrial
   respiration and glycolysis can be rescued by re-expression of an RNAi
   resistant version of hnRNP U at a level proportional to the amount of
   ectopic hnRNP U, ruling out the possibility of an off-target effect in
   the analysis (Supplementary Fig. [303]9b–f). Consistently, hnRNP U
   depletion also reduced cell proliferation (Fig. [304]6f). Since
   depleting hnRNP U increased RG4 stability (Fig. [305]3i, j), we
   wondered whether this effect was also observed after ligand-mediated
   stabilization. Indeed, Supplementary Fig. [306]9g shows that PhenDC3
   reduced glycolysis in three different cell lines, suggesting that RG4
   stabilization induced by either small molecule ligands or hnRNP
   U-silencing may have a dual metabolic effect by inhibiting oxidative
   metabolism, both glycolysis and OXPHOS.

Fig. 6. hnRNP U modulates OXPHOS expression and function.

   [307]Fig. 6
   [308]Open in a new tab

   a Western blot analysis in HCT116 cells treated with control (Ctr), and
   2 different hnRNP U (#1, #2) siRNAs for 72 h. The blot shown is a
   representative result from n > 4 independent experiments. b
   Quantification of the protein levels in (a) normalized to β-Actin and
   plotted relatively to si Ctr condition. Data are presented as mean
   values ± SEM of n = 4 independent experiments, *P < 0.05, **P < 0.01,
   ***P < 0.001, ns: non-significant (two-way ANOVA). c Quantification of
   the Oxygen Consumption Rate (OCR) per cell, measured by a Seahorse
   assay, in HCT116 cells treated with control (si Ctr), hnRNP U (si hnRNP
   U), DDX3X (si DDX3X) or a combination of hnRNP U and DDX3X (si hnRNP U
   + si DDX3X) siRNAs for 48 h. Data are presented as mean values ± SEM of
   n = 3 independent experiments. d Quantification of the Oxygen
   Consumption Rate (OCR) linked to basal and maximal mitochondrial
   respiration, ATP turnover, proton leak, and spare respiratory capacity,
   measured by a Seahorse assay, in HCT116 treated with si Ctr, si hnRNP
   U, si DDX3X or a combination of si hnRNP U + si DDX3X for 48 h. Data
   are presented as mean values ± SEM of n = 3 independent experiments,
   *P < 0.05, **P < 0.01 (two-sided paired t-test). e Quantification of
   the ECAR linked to non-glycolytic acidification, glycolytic capacity
   and glycolytic reserve, measured by a Seahorse assay (glycolysis stress
   test), in HCT116 treated with si Ctr and si hnRNP U for 48 h. Data are
   presented as mean values ± SEM of n = 3 independent experiments,
   P-value = 0.0316 for the non-glycolytic acidification (one-sided paired
   t-test). f Proliferation assay in HCT116 cells treated with si Ctr, si
   hnRNP U#1, si hnRNP U#2 for 48 h. Data are presented as mean
   values ± SEM of n = 3 independent experiments, *P < 0.05, **P < 0.01
   (two-sided paired t-test). For all the panels, Source Data is provided
   as a [309]Source Data file indicating exact P-values for (b, d, f).

hnRNP U, DDX3X and GRSF1 cooperate in the translational regulation of
RG4-containing nuclear-encoded mitochondrial mRNAs

   We then sought to define the molecular mechanism underlying the
   function of hnRNP U in translation regulation of RG4-containing
   nuclear-encoded mitochondrial mRNAs. The observation that hnRNP U and
   DDX3X, 1) interact (Supplementary Data [310]1), in agreement with
   Brannan et al.^[311]80), 2) are both recruited on folded RG4s in OMM
   fractions (Figs. [312]3, [313]4c) have a similar function in
   mitochondrial respiration and glycolysis (Fig. [314]6c, d,
   Supplementary Fig. [315]9a) prompted us to investigate whether hnRNP U
   functions cooperatively with DDX3X to control mitochondrial mRNA
   translation and function. We first studied the molecular determinants
   enabling hnRNP U to interact with the RNA and DDX3X. Consistent with
   the evidence that the RGG (arginine-glycine-glycine) domain is involved
   in protein-RG4^[316]81 and protein-protein interactions^[317]82, we
   found that deletion of this domain in the C-terminal region of hnRNP U
   decreased its interaction with both DDX3X (Fig. [318]7a) and RG4s
   (Fig. [319]7b), whereas DDX3X binding to RG4s was unaffected
   (Fig. [320]7b), suggesting that DDX3X may bind to RG4s independently of
   hnRNP U. If the “bind, unfold, lock” model involving DHX36 and hnRNP
   H/F that we proposed previously^[321]26 also applies here, hnRNP U and
   DDX3X could be recruited to RG4s first, followed by GRSF1, a member of
   the hnRNP H/F family, which binds to unfolded RG4s in the OMM fractions
   (Fig. [322]4c), to maintain them in the unfolded conformation. This
   would agree with data showing that GRSF1 (1) is found in OMM fractions
   (Fig. [323]4b) where it associates with G-rich sequences unable to form
   RG4s (Figs. 4c), (2) it is involved in binding and unwinding G4
   structures^[324]38,[325]83, (3) predominantly binds to nuclear-encoded
   mRNAs^[326]72, specifically to RG4s within 5’ and 3’UTRs^[327]84 and
   (4) regulates mRNA translation via 5’ and 3’UTR
   mechanisms^[328]85–[329]87. Furthermore, this scenario would be
   consistent with findings showing that DDX3X, 1) binds to RG4s in mRNAs
   encoding oxidative phosphorylation proteins^[330]25, (2) plays a role
   in translation when associated with 3’UTRs^[331]88, and (3) impacts on
   mitochondrial functions^[332]69. If the model is true, we would expect
   that the depletion of proteins that resolve RG4s and those that keep
   them unfolded would lead to an increase in RG4 formation. In agreement
   with this, we found that RG4 foci visualized using BioCyTASQ increased
   after DDX3X and GRSF1 silencing (Supplementary Fig. [333]10a,b). Then,
   we performed RIP assays using antibodies against GRSF1 or DDX3X and
   cytoplasmic extracts, which we combined with hnRNP U silencing to
   determine the cooperativity among these factors. We observed that DDX3X
   bound to the same targets as hnRNP U, and, in agreement with
   Fig. [334]7b, that depleting hnRNP U did not alter the binding of DDX3X
   to these targets (Fig. [335]7c, Supplementary Fig. [336]10c). This
   suggests that, as previously demonstrated for the hnRNP H/F RBP and the
   DHX36 helicase^[337]26, DDX3X binds first followed by hnRNP U. This
   possibility was confirmed by RG4 pull-down analysis of hnRNP U from
   extracts depleted or not of DDX3X, showing that in the absence of DDX3X
   the binding of hnRNP U to RG4s was reduced but not that of another
   RG4-binding protein, DHX36 (Fig. [338]7d), suggesting a specific
   recruitment of hnRNP U by DDX3X bound to RG4s. GRSF1 was also
   unaffected by the loss of DDX3X binding (Fig. [339]7d), which is
   consistent with the fact that it is a G-rich-BP but also that it did
   not interact directly with hnRNP U, as shown by co-immunoprecipitation
   analyses (Fig. [340]7a, e) and proteomic analysis of hnRNP U
   interactants (Supplementary Data [341]1). These results, together with
   a trend for GRSF1, similar to DDX3X, to bind to the same hnRNP U
   targets (Supplementary Fig. [342]10d, e), suggest that binding of the
   hnRNP U-DDX3X complex and GRSF1 probably involves other factors to
   facilitate the dynamics of RG4 unfolding.

Fig. 7. hnRNP U cooperates with DDX3X and GRSF1 to regulate RG4-dependent
translation.

   [343]Fig. 7
   [344]Open in a new tab

   a Immunoprecipitation (IP) with the Flag antibody of protein from
   cytoplasmic cell extracts (CE) of HCT116 cells transfected with
   flag-tagged unmodified hnRNP U (WT), hnRNP U RBD deleted (ΔRBD), or
   empty vector (e.v), followed by western blot analysis. b RNA affinity
   chromatography using the RG4 sequences either native (WT),
   7-deaza-modified (7dG) or mutated (Mut) and CE of HCT116 cells
   transfected with plasmids encoding for hnRNP U-Flag WT or ΔRBD,
   followed by western blot analysis. c IP with the DDX3X antibody of
   cellulo RNA-protein complexes (RIP) in CE of HCT116 treated with
   control (Ctr) or hnRNP U (U) siRNAs, followed by RT-qPCR analysis. Fold
   changes are indicated in italic. *P < 0.05, **P < 0.01, ***P < 0.001
   (one-sided paired t-test). d RNA affinity chromatography using the RG4
   sequences either WT, 7dG, and CE of HCT116 treated control (si Ctr) or
   DDX3X (si DDX3X) siRNAs, followed by western blot analysis. e IP with
   the GRSF1 antibody of CE of HCT116 treated or not with benzonase,
   followed by western blot analysis. Shown is a representative result
   from n = 3 independent experiments. f Western blot analysis in HCT116
   cells treated with si Ctr, si hnRNP U, or si DDX3X for 48 h and
   quantification of the protein levels normalized to β-Actin.
   P-value = 0.0067 for hnRNP U protein in si hnRNP U condition (two-sided
   paired t-test). g Western blot analysis in HCT116 cells treated with si
   Ctr, si hnRNP U, si DDX3X or both si hnRNP U + si DDX3X for 72 h. h
   Quantification of the protein levels in (g) normalized to β-Actin.
   *P < 0.05, **P < 0.01, (two-sided paired t-test). In (a, b, c, d, f, g,
   h), source data are provided as a Source Data file indicating exact
   P-values for (c, h). Data are presented as mean values ± SEM of n = 3
   for (f, h), of n = 4 in duplicates for (c) independent experiments. In
   (a, b, d, f, g), the blot shown is a representative result from n = 3
   independent experiments. When indicated, ns non-significant.

   To determine whether the physical interaction between DDX3X and hnRNP U
   resulted in a functional interplay, we first investigated the
   possibility of a cross-regulation between the two proteins and then
   compared the effect of their co-depletion with that of individual hnRNP
   U or DDX3X silencing on mitochondrial protein expression. We found that
   inhibition of hnRNP U did not alter DDX3X expression and vice versa
   (Fig. [345]7f). Individual inhibition of either protein had a similar
   effect on the expression of two OXPHOS proteins (Fig. [346]7g, h) which
   is consistent with the findings that hnRNP U and DDX3X share a subset
   of mitochondrial mRNA targets. The observation that co-silencing of the
   two proteins did not have a significantly greater effect than depletion
   of hnRNP U and DDX3X individually (Fig. [347]7g, h) suggests a
   sequential recruitment model rather than a synergistic one in which
   binding of one would potentiate that of the other. This conclusion is
   also supported by mitochondrial and glycolysis assays after individual
   and co-depletion of hnRNP U and DDX3X (Fig. [348]6c, d, Supplementary
   Fig. [349]9a). Taken together, our results suggest that hnRNP U, DDX3X,
   and GRSF1 are players in RG4-dependent OMM localized translational
   regulation impacting mitochondrial function and energy metabolism.

Discussion

   Gaining insight into mitochondrial protein synthesis is essential to
   better untangle the link between mitochondrial metabolism and cancer
   development and to identify novel vulnerabilities that can be targeted
   for therapeutic purposes^[350]2. Emerging evidences support the notion
   of compartmentalized translation and co-translational targeting, which
   relies on translation factories providing a means of regulating the
   metabolism of nascent proteins where specific mRNA subsets are
   translated^[351]15. One key question to go further in this concept and
   explore its clinical applicability is what are the molecular
   determinants driving this specificity. A number of RBPs have been shown
   to play a role^[352]47,[353]89 but the cis determinants remain poorly
   characterized, with the role of RNA structures not yet proven. We
   propose here that RG4 structures are involved in mitochondrial mRNA
   translation and that their role in protein synthesis, association with
   OMMs, and mitochondrial function depend on their structural dynamics
   that are regulated by hnRNP U and modulated by small-molecule ligands.
   In our model (Fig.[354]8), when the unfolded form is favored
   co-translational localization of nuclear-encoded mitochondrial mRNAs
   occurs at the OMM, allowing mitochondrial proteins to be synthesized
   and imported into the mitochondria, resulting in efficient
   mitochondrial respiration. However, shifting the structural equilibrium
   to a stably folded RG4 conformation, or failing to form an RG4,
   inhibits both translation and cytoplasmic protein expression of
   nuclear-encoded mitochondrial mRNAs at the OMM. This effect is
   associated with the physical uncoupling of the mitochondrial mRNA from
   both the encoded protein and mitochondria, resulting in the inhibition
   of mitochondrial respiration. Although PhenDC3 or hnRNP U silencing
   mostly reduced mRNA translation and expression, we also observed
   examples of translational activation after these treatments
   (Fig. [355]2b, c). These different effects may be explained by the
   interaction between RG4s and other factors regulating translation or
   the competition between ligands and RBPs for RG4-binding^[356]90.

Fig. 8. Model for the role of hnRNP U-RG4 interactions in regulating
OMM-localized mRNA translation of nuclear-encoded mitochondrial mRNAs.

   [357]Fig. 8
   [358]Open in a new tab

   Following the “bind, unfold, lock” model previously proposed^[359]26,
   hnRNP U in complex with DDX3X unfolds RG4s and indirectly recruits
   GRSF1 onto G-rich sequences. This molecular mechanism, which is
   regulated by mTOR, activates OMM-localized mRNA translation of
   nuclear-encoded mitochondrial mRNAs carrying RG4s in their 3’UTR,
   resulting in mitochondrial protein expression. Ultimately, this
   modulates mitochondrial functions, increasing energy metabolism and
   consequently cancer cell proliferation.

   The underlying translational regulatory mechanism, similar to the
   “bind, unfold, lock” model we proposed previously^[360]26, could
   involve DDX3X recruiting hnRNP U to folded RG4s, which in turn
   facilitates their unfolding and makes GRSF1 binding accessible via
   hnRNP U and additional factors that remain to be characterized. Our
   results suggest that this RG4-dependent mechanism may act as an
   effector of oncogenic mTOR signaling, which is known to regulate
   mitochondrial mRNA translation and thereby provide a source of ATP for
   protein synthesis^[361]75. Although the link between RG4s and upstream
   oncogenic signaling requires further investigation, our results showing
   that mTOR inhibition induces global folding of RG4s while reducing the
   binding of hnRNP U to RG4s and polysomes (Fig. [362]5h, i,
   Supplementary Fig. [363]8a, c) indicate the possibility that mTOR
   inhibition induces a reduction in ATP synthesis via inhibition of
   RG4/hnRNP U-dependent mitochondrial mRNA translation and that this ATP
   shortage in turn impacts on the activity of RG4 helicases, including
   DDX3X. This hypothesis is consistent with previous data showing that
   depletion of cellular ATP levels by inhibition of oxidative
   phosphorylation or glycolysis prevented RG4 unfolding during recovery
   from starvation-induced stress^[364]77. One important question that
   needs to be addressed, concerns the mechanism by which RG4s at 3’UTRs
   regulate mRNA translation. One possibility arising from recent ribosome
   profiling data (based on sequencing of mRNA fragments enclosed within
   the translating ribosome), which shows that the translation of the main
   ORF (mORF) is regulated by small ORFs located within 3’UTRs
   (dORFs)^[365]91,[366]92, is that RG4s could be involved in this
   regulatory mechanism, for which the molecular details are still largely
   unexplored. Supporting this hypothesis, RG4s colocalizing with small
   ORFs located upstream of the ORF (uORF) were shown to regulate mORF
   translation^[367]93. In line with this possibility, we found that
   mitochondria-related terms were enriched among the mRNAs in which dORFs
   were experimentally identified^[368]91,[369]92. Moreover, these
   elements are present in one-fourth of the RG4-containing mRNAs bound by
   hnRNP U (Supplementary Fig.11), indicating that RG4s might be involved
   in dORF-mediated regulation of mORF translation. Since RG4
   stabilization, induced by ligands or by depleting hnRNP U, inhibits
   mitochondrial respiration and glycolysis in different cancer cell types
   (Figs. [370]1c, e, and. [371]6c–e, Supplementary Fig. [372]2c, d,
   Supplementary Fig. [373]4b, c, Supplementary [374]9g), the RG4/hnRNP
   U-dependent mechanism described here is not expected to be cancer
   type-specific per se. However, the quantitatively different effects of
   the ligand on mitochondrial respiration, and proportionally on the
   proliferation of the panel of tested cancer cells, correspond to the
   different level of dependency of those cells on mitochondrial
   function^[375]2. This is consistent with the view that cancer cells and
   tumors have heterogeneous metabolic preferences and