Biology subjects

Vagner, S.

Publications and source records attributed to Vagner, S..

5 recordsLinked to original sources

bioRxiv2023

Intronic polyadenylation isoforms in the 5' part of genes constitute a source of microproteins and are involved in cell response to cisplatin

Transcript isoforms generated by intronic polyadenylation (IPA) are widely regulated in various biological processes and often encode protein isoforms. Microproteins are small proteins translated from small open reading frames (sORFs) in noncoding RNAs and mRNAs, but their production by IPA isoforms is unknown. Using 3-seq and long-read RNA-seq analyses in lung cancer cells, we show that cisplatin, a DNA-crosslinking anticancer agent, upregulates IPA isoforms relative to full-length mRNAs in long genes. A subset of cisplatin-regulated IPA isoforms are poorly associated with heavy polysomes and terminate upstream of the annotated translation initiation codon of genes. Such IPA isoforms in the PHF20 and PRKAR1B genes are associated with light polysomes, contain Ribo-Seq-supported sORFs in an alternative last exon within the annotated 5UTR part of genes, and are translated into microproteins. For PRKAR1B, the microprotein was detected by Western blot and immunofluorescence after transfection of a tagged isoform; and siRNA depletion of the endogenous IPA isoform, CRISPR deletion of the IPA site, or CRISPR mutation of the sORF initiation codon led to increased cell survival to cisplatin. Based on Ribo-Seq and mass-spectrometry data sets, we identified 156 genes producing both a canonical protein-coding mRNA and a microprotein-coding 5UTR-located IPA isoform (coined miP-5UTR-IPA isoform) regulated by cisplatin. Finally, the regulation of (miP-5UTR-)IPA versus full-length isoforms by cisplatin involved an inhibition of transcription processivity in a FANCD2 and senataxin-dependent manner. Altogether, these findings reveal the novel paradigm of miP-5UTR-IPA genes and their role in cancer cell response to a genotoxic agent. HIGHLIGHTS- Cisplatin increases intronic-polyadenylation versus full-length transcript isoforms in long genes through a FANCD2 and senataxin-dependent decrease of transcription processivity - A subset of cisplatin-regulated intronic-polyadenylation isoforms terminate in the annotated 5UTR part of genes and encode microproteins, thus we coined them miP-5UTR-IPA isoforms - The miP-5UTR-IPA isoform of PRKAR1B impacts cisplatin sensitivity and its effect is mediated by its small ORF - We identify 156 genes producing both a canonical protein-coding mRNA and a microprotein-coding miP-5UTR-IPA transcript

molecular biology↗

bioRxiv2023

53BP1 interacts with the RNA primer from Okazaki fragments to support their processing during unperturbed DNA replication

RNA-binding proteins are found at replication forks, but their direct interaction with DNA-embedded RNA species that inevitably shape physiological DNA replication remains unexplored. Here we report that 53BP1, involved in the DNA damage and replication stress response, is an RNA-binding protein that directly interacts with Okazaki fragments, in the absence of any external stress. The bulk chromatin association of 53BP1 shows dramatic dependence on PRIM1, which synthesizes the RNA primer of Okazaki fragments. The direct recruitment of 53BP1 to nascent DNA shows susceptibility to in situ ribonuclease A treatment. Conversely, depletion of FEN1, which results in the accumulation of uncleaved RNA primers, leads to an upregulation of 53BP1 levels at the replication forks, suggesting that RNA primers contribute to the recruitment of 53BP1 at the lagging DNA strand. 53BP1 depletion induces an accumulation of S phase poly(ADP-ribose), which constitutes a sensor of unligated Okazaki fragments. Collectively, our data indicate that 53BP1, distinct from its canonical mode of chromatin-binding, is anchored at the replication fork through its RNA-binding activity, highlighting the role of an RNA-protein interaction at DNA replication forks.

molecular biology↗

bioRxiv2023

MET functions in tumour progression and therapy resistance are repressed by intronic polyadenylation

Intronic polyadenylation (IPA) leads to the production of transcript isoforms with alternative last exons in thousands of mammalian genes. Widespread regulation of IPA isoforms was observed during oncogenic transformation and in tumours versus healthy tissues, and several IPA isoforms were involved in oncogenesis. However, little is known about the potential involvement of IPA in tumour progression, such as cancer cell invasiveness and metastasis, and in resistance to anticancer therapies. Here, we show that an IPA isoform of MET (short MET) whose production is inhibited by U1 snRNP (U1), an essential ribonucleoprotein complex that recognizes the 5 exon-intron junction of pre-mRNA, is associated with better prognosis in breast cancer. Induction of the short MET isoform, using a steric-blocking antisense oligonucleotide targeting the U1 binding site in the vicinity of the short MET alternative polyadenylation site, antagonizes cell invasiveness. U1 blockade with an antisense oligonucleotide targeting the U1 snRNA also decreases breast cancer cell invasiveness, in both human and mouse cancer cell models, and this effect involves IPA induction in MET and several genes belonging to the RAS/RAF/MAPK signalling pathway. Finally, short MET relieves melanoma cell resistance to MAPK cascade-targeted therapy in vitro and in vivo. IPA isoform levels of MET and a few other genes (mTOR, EGFR and CTNNA1) help predict such resistance in patients. Altogether, our findings provide evidence for a role of IPA in both cancer cell invasiveness and resistance to therapy. This suggests that IPA isoforms can be exploited as biomarkers and therapeutic targets to combat tumour progression.

molecular biology↗

bioRxiv2022

RGG-motif proteins regulate mRNA translation upon genotoxic stress

Genotoxic stress response (GSR) mediated by mRNA translation and decay regulation remains poorly explored. Here, we identify a unique role of yeast RGG-motif protein Scd6 and its human ortholog LSM14A in mRNA translation control upon hydroxyurea (HU)-mediated GSR. Scd6/LSM14A, but not all tested RGG-containing proteins, localize to HU-induced cytoplasmic puncta in an RGG-dependent manner. The absence of Scd6 increases HU tolerance but sensitizes the cells to HU upon overexpression of SRS2, a known dampener of the DNA- damage response. Scd6 binds SRS2 mRNA to repress its translation in cytoplasmic granules upon HU stress. Scd6-SRS2 interaction is modulated by arginine methylation (AM) and the LSm-domain, which acts as a cis-regulator of Scd6 AM. Polysome-profiling experiments indicate that LSM14A regulates the translation of NHEJ factor mRNAs such as LIG4 (DNL4 homolog) and RTEL1 (SRS2 functional homolog), and the NHEJ activity in response to HU. Overall, this report unveils the role of AM and Scd6/LSM14A in the GSR by determining the translation status of specific mRNAs.

cell biology↗

bioRxiv2022

Uncoupling from transcription protects polyadenylation site cleavage from inhibition by DNA damage

Pre-mRNA 3-end processing by cleavage and polyadenylation (CPA) is a nuclear process in which RNA polymerase II (Pol II) transcripts are cleaved at the polyadenylation site (PAS cleavage) before addition of a poly(A) tail. While PAS cleavage is usually coupled to transcription termination, for some pre-mRNAs it occurs post-transcriptionally, i.e. after pre-mRNA release from chromatin to nucleoplasm through a downstream co-transcriptional cleavage (CoTC) event. DNA-damaging agents such as ultraviolet-C (UV) irradiation trigger rapid shutdown of pre-mRNA 3-end processing. However, specific compensatory mechanisms exist to ensure efficient 3-end processing for some pre-mRNAs encoding proteins involved in the DNA damage response (DDR), such as the p53 tumor suppressor protein. Here, we show that PAS cleavage of the p53 pre-mRNA occurs in part post-transcriptionally, in a PCF11-independent manner, in the nucleoplasm, following a CoTC-type event. Upon UV-irradiation, cells with an engineered deletion of the p53 CoTC site exhibit impaired 3-end processing of the p53 pre-mRNA, decreased mRNA and protein levels of p53 and its transcriptional target, p21, and altered cell cycle progression. Finally, using a transcriptome-wide analysis of PAS cleavage, we identified additional-including DDR related-pre-mRNAs whose PAS cleavage is maintained in response to UV and occurs post-transcriptionally. These findings indicate that CoTC-type cleavage of pre-mRNAs, followed by PAS cleavage in the nucleoplasm, allows specific pre-mRNAs to escape 3-end processing inhibition in response to UV-induced DNA damage.

molecular biology↗