Alternative cleavage and polyadenylation generates downstream uncapped RNA isoforms with translation potential

Malka, Yuval; Alkan, Ferhat; Ju, Shinyeong; Körner, Pierre-René; Pataskar, Abhijeet; Shulman, Eldad D.; Loayza‐Puch, Fabricio; Champagne, Julien; Wenzel, Casper; Faller, William J.; Elkon, Ran; Lee, Cheolju; Agami, Reuven

doi:10.1016/j.molcel.2022.09.036

Cited by 13 publications

(14 citation statements)

References 77 publications

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“…The 3’-RNA capping and pulldown (3’-PD) data in U2OS cells ( 72, 73 ) were downloaded from Gene Expression Omnibus under the accession number GSE84068 including three 3’-PD replicas (GSM2226722–GSM2226724) and three total polyA + RNA-seq replicas for normalization (GSM2226713–GSM2226715). The per-bin coverage around 5’-splice sites was computed as for Cleave-seq.…”

Section: Methodsmentioning

confidence: 99%

“…The per-bin cleave-seq signal was computed around 5'-splice sites using deeptools computeMatrix tool with the following parameters computeMatrix reference-point -a 150 -b 20 -bs 5 --nanAfterEnd --missingDataAsZero --skipZeros and consequently averaged between replicas and introns for visualization. The 3'-RNA capping and pulldown (3'-PD) data in U2OS cells (72,73) were downloaded from Gene Expression Omnibus under the accession number GSE84068 including three 3'-PD replicas (GSM2226722-GSM2226724) and three total polyA + RNA-seq replicas for normalization (GSM2226713-GSM2226715). The per-bin coverage around 5'-splice sites was computed as for Cleave-seq.…”

Section: Splicing Metricsmentioning

confidence: 99%

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Transcriptome sequencing suggests that pre-mRNA splicing counteracts widespread intronic cleavage and polyadenylation

Vlasenok

Margasyuk

Pervouchine

2022

Preprint

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Alternative splicing (AS) and alternative polyadenylation (APA) are two crucial steps in the post-transcriptional regulation of eukaryotic gene expression. Protocols capturing and sequencing RNA 3'-ends have uncovered widespread intronic polyadenylation (IPA) in physiological and disease conditions, where it is currently attributed to stochastic variations in pre-mRNA processing. Here, we took advantage of the massive amount of RNA-seq data generated by the Genotype Tissue Expression project (GTEx) to simultaneously identify and match tissue-specific usage of intronic polyadenylation sites with tissue-specific splicing. A combination of computational methods including the analysis of short reads with non-templated adenines confirmed highly abundant IPA events. Among them, composite terminal exons and skipped terminal exons expectedly correlate with splicing, however we also observed a considerable fraction of IPA events that lack AS support and can be attributed to lariat polyadenylation (LPA). We hypothesize that LPA originates from a dynamic coupling between APA and AS, in which the spliceosome removes an intron after CPA have already occurred in it. Taken together, these results suggest that co-transcriptional pre-mRNA splicing could serve as a natural mechanism of suppression of premature transcription termination.

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Section: Methodsmentioning

confidence: 99%

Section: Splicing Metricsmentioning

confidence: 99%

Transcriptome sequencing suggests that pre-mRNA splicing counteracts widespread intronic cleavage and polyadenylation

Vlasenok

Margasyuk

Pervouchine

2022

Preprint

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“…We have previously shown that endonuclease cleavage of RNA at an alternative polyadenylation site can generate uncapped, autonomous RNA fragments with translational potential 2,7 . This process requires several features, including APA-mediated cleavage, structured RNA to stabilize the new 5'-end, and m6A modifications to facilitate cap-independent translation.…”

Section: Dynamic Expression Of Protein Domains Through Rna Dicingmentioning

confidence: 99%

RNA dicing regulates the expression of an oncogenic JAK1 isoform

Malka,

van der Kammen,

et al. 2024

Preprint

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AbstractmRNA transcripts have limited potential for protein synthesis, defined by their open reading frames1. However, recent advances have revealed a far more complex reality, in which the proteome exceeds the perceived limits of the transcriptome2–6exposing a significant gap in our understanding. Our prior studies have demonstrated that mRNA can undergo further processing, yielding truncated, uncapped mRNAs with translation potential2,7. Yet, the biological importance of this process remains mostly unclear. Here, we demonstrate that cleavage within the coding sequence of JAK1 mRNA produces an uncapped variant downstream to the cleavage site, at the expense of the full-length transcript. This results in the independent translation of the JH1 kinase domain, a process we term ‘RNA dicing’. Notably, canonical and diced JAK1 variants have distinct impacts on cell proliferation and tumorigenesis, operating independently, localizing to distinct cellular compartments. In addition, the activation of JAK1 signaling through IFNγ induction promotes the dicing of JAK1, thereby altering the balance of isoforms present. Base editor screens reveal that stop-codon nonsense mutations, which are typically considered loss-of-function, have differing impacts depending on their position relative to the dicing site. In agreement with this, JAK1 nonsense frameshift (JAK1fs) mutations in endometrial tumors inhibit the tumor-suppressive functions of canonical JAK1, while amplifying the oncogenic potential of the diced JH1 kinase domain. Lastly, we demonstrate that Momelotinib8, a JAK1-specific inhibitor, is more effective in cancer cells carrying a “loss-of-function” JAK1fs mutation, highlighting the significant impact of RNA dicing biology as a potent tool for patient stratification. Our findings characterize RNA dicing as a fundamental regulatory machinery that diversifies the potential products of a single mRNA molecule, and allows for significant variation in biological function.

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“…On the other hand, sncRNAs can also be generated by cleavage of other small RNAs, as illustrated by the discovery of stable small fragments of tRNAs [ 41 ] and other structural RNAs [ 40 ]. Overall, it appears that post-transcriptional cleavage is a wide-spread phenomenon that can explain the generation of multiple long and small RNA species [ 42 , 43 , 44 , 45 ], as reviewed in [ 40 ]. In fact, the genomic overlap between unannotated long and small RNAs has led to a hypothesis that a significant fraction of the lncRNA transcriptome functions as a precursor to sncRNAs [ 7 ].…”

Section: Introductionmentioning

confidence: 99%

Evidence for Existence of Multiple Functional Human Small RNAs Derived from Transcripts of Protein-Coding Genes

Gao

Wang

Cao

et al. 2023

IJMS

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The human genome encodes a multitude of different noncoding transcripts that have been traditionally separated on the basis of their lengths into long (>200 nt) or small (<200 nt) noncoding RNAs. The functions, mechanisms of action, and biological relevance of the vast majority of both long and short noncoding transcripts remain unknown. However, according to the functional understanding of the known classes of long and small noncoding RNAs (sncRNAs) that have been shown to play crucial roles in multiple biological processes, it is generally assumed that many unannotated long and small transcripts participate in important cellular functions as well. Nevertheless, direct evidence of functionality is lacking for most noncoding transcripts, especially for sncRNAs that are often dismissed as stable degradation products of longer RNAs. Here, we developed a high-throughput assay to test the functionality of sncRNAs by overexpressing them in human cells. Surprisingly, we found that a significant fraction (>40%) of unannotated sncRNAs appear to have biological relevance. Furthermore, contrary to the expectation, the potentially functional transcripts are not highly abundant and can be derived from protein-coding mRNAs. These results strongly suggest that the small noncoding transcriptome can harbor multiple functional transcripts that warrant future studies.

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Alternative cleavage and polyadenylation generates downstream uncapped RNA isoforms with translation potential

Cited by 13 publications

References 77 publications

Transcriptome sequencing suggests that pre-mRNA splicing counteracts widespread intronic cleavage and polyadenylation

Transcriptome sequencing suggests that pre-mRNA splicing counteracts widespread intronic cleavage and polyadenylation

RNA dicing regulates the expression of an oncogenic JAK1 isoform

Evidence for Existence of Multiple Functional Human Small RNAs Derived from Transcripts of Protein-Coding Genes

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