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

Yuval Malka*, Ferhat Alkan, Shinyeong Ju, Pierre Rene Körner, Abhijeet Pataskar, Eldad Shulman, Fabricio Loayza-Puch, Julien Champagne, Casper Wenzel, William James Faller, Ran Elkon, Cheolju Lee, Reuven Agami*

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

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Abstract

The use of alternative promoters, splicing, and cleavage and polyadenylation (APA) generates mRNA isoforms that expand the diversity and complexity of the transcriptome. Here, we uncovered thousands of previously undescribed 5' uncapped and polyadenylated transcripts (5' UPTs). We show that these transcripts resist exonucleases due to a highly structured RNA and N6-methyladenosine modification at their 5' termini. 5' UPTs appear downstream of APA sites within their host genes and are induced upon APA activation. Strong enrichment in polysomal RNA fractions indicates 5' UPT translational potential. Indeed, APA promotes downstream translation initiation, non-canonical protein output, and consistent changes to peptide presentation at the cell surface. Lastly, we demonstrate the biological importance of 5' UPTs using Bcl2, a prominent anti-apoptotic gene whose entire coding sequence is a 5' UPT generated from 5' UTR-embedded APA sites. Thus, APA is not only accountable for terminating transcripts, but also for generating downstream uncapped RNAs with translation potential and biological impact.

Original languageEnglish
Pages (from-to)3840-3855
Number of pages16
JournalMolecular Cell
Volume82
Issue number20
DOIs
Publication statusPublished - 20 Oct 2022

Bibliographical note

Funding Information:
R.A. is supported by the Dutch Cancer Society ( KWF projects 13647, 11574) and the European Research Council (EEG- CEC / EU 832844). A.P. is supported by a long-term EMBO fellowship grant ( EMBO ALTF 796-2018). S.J. and C.L. are supported by the National Research Foundation of Korea (grant number 2020R1A2C2003685). We thank Eran Rosenthal and Tommy Kaplan for technical assistance with the HMM model, Ittai Ben-Porath for comments on the manuscript, and Ron Kerkhoven and the Netherlands Cancer Institute (NKI- AVL ) Genomics Core Facility in sequencing. We thank F. Van Gemert and H. te Riele for providing Bcl2 overexpression retrovirus.

Funding Information:
Next, we set to explore the most influential biogenesis pathway leading to the production of 5′ UPTs. Based on our observations, we postulated that 5′ UPTs are being generated through endonuclease activity. Previous work in mammalian cells produced widespread evidence for endonuclease cleavage events, but further investigation failed to connect them to Drosha, Dicer, and RNA-induced silencing complex (RISC) (Karginov et al., 2010). In addition, transcriptomic capped analysis of gene expression (CAGE) revealed widespread intra-exonoic capping that did not arise from conventional transcription initiation (Mercer et al., 2010, 2011). Interestingly, a consensus motif search analysis around TrPt sites identified A(A/T)TAAA (6.3 × 10−677), a conserved polyadenylation signal (PAS) motif (Figure 4A), as the top hit. The PAS motif was enriched upstream of TrPts in CDSs, indicating that APA is likely a prominent upstream factor in the generation of 5′ UPTs (Figure 4B). To directly address this possibility, we performed 3′ end sequencing (3′ seq) of mRNAs that captures polyadenylation sites of mRNAs. Bioinformatics analysis of TrPt sites (11,052 genes with CDSs or 3′ UTR TrPts) showed a 29.1% overlap with active polyadenylation sites within +/−100-bp window, further supporting an overall correlation of 3′ ends with the identified TrPts (Figure 4C and Figure S5A), implicating the endo-nucleolytic cleavage activity at APA sites as a major principal event generating 5′ UPTs downstream of TrPts.To experimentally evaluate the impact of APA usage on 5′ UPT biogenesis, we performed a series of knockdowns of the most prominent positive and negative APA regulators. Loss of either PCF11 or INTS11 (an APA termination factor and the endonuclease factor of integrator I, respectively) was recently shown to inhibit APA and promote 3′ UTR lengthening (Dasilva et al., 2021; Wang et al., 2019). We therefore knocked down PCF11 and INTS11 using small interfering RNA (siRNA) in HeLa cells (Figure S5B) and performed HMM and Z score analyses for each condition in TEX control and treated samples ( Tables S13–S15 for control, PCF11KD, and INTS11KD, respectively, and Figure S5C–E). Figure S5D shows that PCF11KD or INTS11KD substantially reduced 5′ UPT expression. In parallel, we investigated the causal role of two of the most prominent nuclear APA factors, U1 and NUDT21, in the generation and translatability of 5′ UPTs. We used antisense morpholino oligos and siRNAs to knock down U1 and NUDT21 (Figure S5G) in HeLa cells (Oh et al., 2020). RNA-seq expression analysis ( Tables S16–S17, respectively) revealed vast expression of truncated downstream-mRNA variants in both U1KD and NUDT21KD cells (Figures 4D and 4E). To link these mRNA variants with 5′ UPTs, we treated RNA extraction from each condition with TEX and analyzed by RNA-seq. We found increased TEX sensitivity by both U1KD and NUDT21KD (4,917 and 5,407 genes with TrPts in control morpholino and U1KD, respectively [ Tables S18–S19], 5,930 and 6,537 and genes for control siRNA and NUDT21KD, respectively [ Tables S20–S21]), supporting APA usage as a significant driver for 5′ UPTs biogenesis (Figure S5H–K). Comparative analysis of these data further substantiated this notion and showed higher sensitivity to TEX treatment in U1KD and NUDT21KD compared with their corresponding controls (Figure S5L–M).Since 5′ UPT expression is associated with strong RNA structure at the 5′ end (Figures 2D and 2E), we wished to determine the RNA structure at the 5′ end of Bcl2 5′ UPT. In Figure S6E, we show that the 5′ end of Bcl2 5′ UPT is a highly structured region that potentially can provide protection from endonuclease digestion (Lorenz et al., 2011). In addition, the sequence of the first 10 nt of this 5′ UPT predicted higher folding energy when compared with shuffled sequences that have the same mono-, di-, and tri-nucleotide frequencies (1,000 shuffles for each) (Figure S6F, top panel). Moreover, the first 10-nt region seems to have the highest opening energy when compared with other 10-nt regions on the input RNA sequence (Figure S6F, middle panel). A similar observation was made when these assays were repeated with different RNA lengths (80, 100, and 164 nts). Moreover, single nucleotide polymorphisms (SNPs) appearing in this region are unlikely to disrupt its RNA structure (Figure S6F, bottom panel). Thus, a strong secondary structure is suggested to support Bcl2-5’ UPT expression.Interestingly, recent studies described a strong link between APA and m6A modification. For example, m6A-containing transcripts are subjected to a higher APA usage (Molinie et al., 2016; Yue et al., 2018). In addition, the m6A reader YTHDC1 is associated with CPSF6, a 3′ end polyadenylation factor, and loss of YTHDC1 leads to extensive alternative polyadenylation in oocytes (Kasowitz et al., 2018). Thus, APA usage is linked to m6A modification and supports 5′ UPTs biogenesis.R.A. is supported by the Dutch Cancer Society (KWF projects 13647, 11574) and the European Research Council (EEG-CEC/EU 832844). A.P. is supported by a long-term EMBO fellowship grant (EMBO ALTF 796-2018). S.J. and C.L. are supported by the National Research Foundation of Korea (grant number 2020R1A2C2003685). We thank Eran Rosenthal and Tommy Kaplan for technical assistance with the HMM model, Ittai Ben-Porath for comments on the manuscript, and Ron Kerkhoven and the Netherlands Cancer Institute (NKI-AVL) Genomics Core Facility in sequencing. We thank F. Van Gemert and H. te Riele for providing Bcl2 overexpression retrovirus. Y.M. conceived the project, designed and performed experiments, analyzed data, and wrote the manuscript; R.A. conceived the project, wrote the manuscript, and supervised the project; P.-R.K. A.P. F.A. E.S. W.J.F. and R.E. analyzed data; S.J. and C.L. performed mass spectrometry; F.L.-P. performed ribosome profiling; J.C. performed T cell killing assay; C.W. performed experiments. All authors read and approved the manuscript. The authors declare no competing interests.

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