RNA Ligation Precedes the Retrotransposition of U6/LINE-1 Chimeric RNA

RNA ligation precedes the retrotransposition of U6/LINE-1 chimeric RNA

John B. Moldovana,1, Yifan Wanga,b, Stewart Shumanc, Ryan E. Millsa,b, and John V. Morana,d,1

aDepartment of Human Genetics, University of Michigan Medical School, Ann Arbor, MI 48109; bDepartment of Computational Medicine and Bioinformatics, University of Michigan Medical School, Ann Arbor, MI 48109; cMolecular Biology Program, Sloan Kettering Institute, New York, NY 10065; and dDepartment of Internal Medicine, University of Michigan Medical School, Ann Arbor, MI 48109

Edited by Marlene Belfort, University at Albany, Albany, NY, and approved August 29, 2019 (received for review March 28, 2018) Long interspersed element-1 (LINE-1 or L1) amplifies via retrotrans- phosphate (45). The 2′,3′-cyclic phosphate is generated post- position. Active L1s encode 2 proteins (ORF1p and ORF2p) that bind transcriptionally by the Mpn1 enzyme (46, 47), which is encoded their encoding transcript to promote retrotransposition in cis.The by the U6 snRNA biogenesis phosphodiesterase 1 (USB1)gene. L1-encoded proteins also promote the retrotransposition of small- Deletions or mutations in USB1 disrupt U6 snRNA 3′ end pro- interspersed element RNAs, noncoding RNAs, and messenger RNAs cessing (46, 47) and are associated with the human genetic disease in trans. Some L1-mediated retrotransposition events consist of poikiloderma with neutropenia (48). a copy of U6 RNA conjoined to a variably 5′-truncated L1, but how The L1 proteins can act in trans to promote the retrotrans- U6/L1 chimeras are formed requires elucidation. Here, we report the position of a variety of cellular RNAs, including, small inter- following: The RNA ligase RtcB can join U6 RNAs ending in a 2′,3′- spersed element (SINE) RNAs (49–52), noncoding RNAs ′ cyclic phosphate to L1 RNAs containing a 5 -OH in vitro; depletion of (53–56), and messenger RNAs (30, 31). L1-mediated retro- endogenous RtcB in HeLa cell extracts reduces U6/L1 RNA ligation effi- transposition events have likely dispersed hundreds of copies ciency; retrotransposition of U6/L1 RNAs leads to U6/L1 pseudogene of U6 snRNA throughout the human genome (57). Approximately formation; and a unique cohort of U6/L1 chimeric RNAs are pre- 100 to 200 U6 pseudogenes consist of a copy of U6 fused to either sent in multiple human cell lines. Thus, these data suggest that avariably5′-truncated L1 or a complementary DNA (cDNA) U6 small nuclear RNA (snRNA) and RtcB participate in the formation derived from a cellular RNA (53, 55–58), and they contain GENETICS of chimeric RNAs and that retrotransposition of chimeric RNA con- structural hallmarks that indicate they were formed by L1 retro- tributes to interindividual genetic variation. transposition [e.g., they end in a poly(A) tail, integrate into an L1 LINE-1 | retrotransposon | U6 snRNA | RtcB | RNA ligation EN consensus cleavage sequence, and are flanked by short, variably sized target-site duplications] (53, 55, 56, 58). Experiments using engineered human L1s suggest that U6/L1 chimeric ong interspersed element-1 sequences (LINE-1s or L1s) com- pseudogenes account for up to 1 out of 15 L1 retrotransposition prise ∼17% of human DNA and have mobilized by a replicative L events in HeLa cells (55) whereas computational analyses process termed retrotransposition (1, 2). Most L1s are immobile strongly suggest that 3 different LINE clades (LINE-1, LINE-2, and (3–5);however,anaveragehumangenomeharbors∼80 to 100 RTE) participate in U6/LINE chimeric pseudogene formation (57). retrotransposition-competent L1s (RC-L1s) (6–8). Human RC-L1s are ∼6 kilobases (kb) and contain a 5′ untranslated region (UTR) that is followed by 2 open reading frames (ORFs) (ORF1 and Significance ORF2) and a short 3′ UTR that ends in a variable length poly- adenosine [poly(A)] tract (4, 9). ORF1 encodes an ∼40-kDa Long interspersed element-1s (L1s) encode 2 proteins (ORF1p nucleic acid binding protein (ORF1p) (10–14) that has nucleic and ORF2p) that preferentially mobilize (i.e., retrotranspose) acid chaperone activity (14, 15). ORF2 encodes an ∼150-kDa their encoding messenger RNA (mRNA) transcript. ORF1p and/or protein (ORF2p) (16–18) that has DNA endonuclease (L1 EN) ORF2p can also mobilize other cellular RNAs, including short (19) and reverse transcriptase (L1 RT) (20, 21) activities. ORF1p, interspersed elements (SINEs), U6 small nuclear RNA (snRNA), ORF2p, and full-length polyadenylated L1 RNA are required for and mRNAs. Here, we demonstrate the RNA ligase RtcB can join efficient L1 retrotransposition in cis (19, 22, 23). U6 snRNA to L1 or other cellular RNAs to create chimeric RNAs; L1 retrotransposition begins with the transcription of a full- retrotransposition of the resultant chimeric RNAs leads to chi- length genomic L1 from an RNA polymerase II promoter that meric pseudogene formation; and chimeric U6/L1 RNAs are part of the transcriptome in multiple human cells. These data sug- resides within its 5′ UTR (24–26). The bicistronic L1 messenger gest RNA ligation contributes to the plasticity of the tran- RNA (mRNA) is exported to the cytoplasm where it undergoes scriptome and that the retrotransposition of chimeric RNAs can translation (27–29). ORF1p and ORF2p preferentially bind their generate genetic variation in the human genome. encoding L1 mRNA by a process termed cis-preference (30, 31),

leading to the formation of an L1 ribonucleoprotein particle Author contributions: J.B.M., Y.W., S.S., R.E.M., and J.V.M. designed research; J.B.M. and (RNP) (10, 13, 17, 32, 33). The association of ORF2p with the Y.W. performed research; J.B.M., Y.W., S.S., and R.E.M. contributed new reagents/analytic L1 mRNA poly(A) tail is a critical step in both L1 RNP for- tools; J.B.M., Y.W., S.S., R.E.M., and J.V.M. analyzed data; and J.B.M., Y.W., S.S., R.E.M., mation and L1 retrotransposition (23). Components of the L1 and J.V.M. wrote the paper. RNP then enter the nucleus where a new L1 copy is integrated Conflict of interest statement: J.V.M. is an inventor on patent US6150160, is a paid con- sultant for Gilead Sciences and a privately held company founded by Flagship Pioneering, into genomic DNA by target site primed reverse transcription and is on the American Society of Human Genetics Board of Directors. (TPRT) (19, 34, 35). This article is a PNAS Direct Submission. U6 small nuclear RNA (snRNA) is a uridine-rich small non- This open access article is distributed under Creative Commons Attribution-NonCommercial- coding RNA that plays an essential role in nuclear intron splicing NoDerivatives License 4.0 (CC BY-NC-ND). – (36 38). U6 snRNA is the most conserved spliceosomal snRNA 1To whom correspondence may be addressed. Email: [email protected] or moranj@ (39), is transcribed by RNA polymerase III (40), and has structural umich.edu. and functional similarities to domain V of self-splicing group II This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. introns (41–44). The major form of U6 snRNA terminates in a 5- 1073/pnas.1805404116/-/DCSupplemental. base poly-uridine [poly(U)] tract that ends in a terminal 2′,3′-cyclic

www.pnas.org/cgi/doi/10.1073/pnas.1805404116 PNAS Latest Articles | 1of11 Downloaded by guest on September 24, 2021 Thus, U6/LINE chimeric pseudogene formation appears to be an to a 5′-truncated L1 also were recovered from these experiments. ancient and ongoing process. These cDNAs sometimes exhibited microhomologies of 1 to 3 Here, we used genetic, molecular biological, biochemical, and nucleotides (nt) at the U6/L1 cDNA junction and were similar computational approaches to dissect the mechanism of U6/L1 in structure to previously described artifacts encountered in RT- chimeric pseudogene formation. We demonstrate that U6/L1 PCR experiments (SI Appendix,Fig.S1C) (64). Thus, U6/L1 chimeric RNAs arise independently of L1 retrotransposition and chimeric RNAs are generated in transfected HeLa cells indepen- are formed through the ligation of a 2′,3′-cyclic phosphate on the dently of L1 retrotransposition. 3′ end of U6 snRNA and 5′-OH on L1 RNA. Biochemical and genetic evidence suggest that the RNA 2′,3′-cyclic phosphate Purified RtcB Ligates U6 RNA to L1 RNA In Vitro. We next investi- ligase RtcB (59–61) can join U6 RNAs ending in a 2′,3′-cyclic gated the mechanism of U6/L1 chimeric RNA formation. The 3′ phosphate to both L1 and other mRNAs (e.g., green fluorescent end of mature U6 snRNA terminates in a 2′,3′-cyclic phosphate protein [GFP] RNAs) containing a 5′-OH. Finally, we dem- (38, 45). During transfer RNA (tRNA) splicing, the tRNA onstrate that U6/L1 chimeric RNAs are a component of the splicing endoribonuclease excises an intron from a subset of transcriptome in multiple human cell lines. tRNA precursor RNAs, generating a 2′,3′-cyclic phosphate and a 5′-OH on the 3′ and 5′ ends of the cleaved tRNA halves, re- Results spectively (65). In archaea and animals, the cleaved tRNAs are U6/L1 RNA Is Generated Independently of L1 Retrotransposition. spliced together by the RNA 2′,3′-cyclic phosphate ligase RtcB Previous hypotheses suggested that U6/L1 chimeric pseudogene (59–61). RtcB joins 2′,3′-cyclic-PO4 and 5′-OH ends via RNA-3′-PO4 formation could occur if ORF2p undergoes a template-switching and RNA-(3′)pp(5′)G intermediates (66, 67). event from L1 RNA to the 3′ end of U6 snRNA during TPRT (53, We hypothesized RtcB could ligate U6 snRNA to L1 RNA to 55). However, recent studies have demonstrated that ORF2p ex- generate U6/L1 chimeric RNAs. To test this hypothesis, we used hibits a profound preference for binding, either directly or in- ribozymes to generate a synthetic human U6 snRNA bearing a directly, to the 3′ poly(A) tract of L1, Alu, and other cellular 2′,3′-cyclic phosphate (herein called U6 > P) and a synthetic L1 RNAs (23, 62), raising the question of how ORF2p would switch RNA fragment containing a 5′-OH (herein called OH-L1) (SI templates to an RNA (e.g., U6 snRNA) that ends in a poly(U) Appendix, Fig. S2 A and B and Methods). We then used a cDNA tract. We hypothesized that U6 snRNA could be joined to L1 oligonucleotide as a splint to ensure that the U6 snRNA 2′,3′- RNA to form a chimeric U6/L1 RNA prior to retrotransposition. cyclic phosphate and L1 5′-OH ends remained in close proximity To test the above hypothesis, HeLa-JVM cells were transfected to one another (Fig. 2A). We reasoned that the DNA oligonu- with either a wild-type engineered human L1 expression plasmid cleotide splint would simulate the situation encountered during (Fig. 1A) (pJM101/L1.3Δneo) or human L1 expression plasmids tRNA splicing where base pairing interactions within and be- that contain a nonsense mutation in ORF1 (Fig. 1A) (pJM108/ tween the tRNA halves stabilize the cleaved tRNA. The resul- L1.3Δneo) or missense mutations in the L1 EN and/or L1 RT tant RNA/DNA complex was incubated with purified bacterial domain of ORF2p that severely inhibit L1 retrotransposition (Fig. RtcB (59) and treated with DNase I to remove the oligonucle- 1A)(pJBM119/L1.3Δneo or pJM105/L1.3Δneo) (19, 22, 31). otide splint. The RNAs were purified, and nested RT-PCR was Whole cell RNAs from transfected HeLa cells were subjected to used to detect U6/L1 chimeric RNA (Fig. 2 A and B). cDNA synthesis using an oligo-dT primer, and the resultant cDNAs Agarose gel analyses revealed that reactions containing the U6 > were used as templates in nested reverse-transcriptase PCRs (RT- P and OH-L1 templates yielded the predicted 305-bp U6/L1 PCRs) using primers complementary to U6 snRNA and the 3′ end of cDNA product (Fig. 2C, lane 3), which was not visible in negative the engineered L1 plasmid (Fig. 1B). To ensure specificity, the outer controls (Fig. 2C, lanes 1 and 2). DNA sequencing revealed that L1 primer (Fig. 1B) (SV40as) was complementary to a specific se- 39 of 46 (∼85%) U6/L1 cDNA products contained a copy of the quence within the engineered L1 expression construct. The RT-PCR 3′ end of a U6 snRNA cDNA ending in 4 thymidine nucleotides products were separated on agarose gels, visible DNA fragments precisely conjoined to the 5′-OH of the full-length L1 RNA were isolated from gels, and the products were characterized using fragment (Fig. 2D). Three products (∼7%) contained a copy of Sanger sequencing (Fig. 1C). RT-PCR products were not detected in the 3′ end of a U6 snRNA cDNA ending in 4 thymidine nucle- control RT-PCR experiments that lacked the reverse transcrip- otides conjoined to a 5′-truncated L1 fragment. These products tase (SI Appendix, Fig. S1A). could arise from the ligation of U6 > P to broken or degraded Sequence analyses revealed the presence of U6/L1 chimeric L1 RNA fragments containing a 5′-OH (Fig. 2D and SI Appen- cDNAs in HeLa cells transfected with either the wild-type or dix, Table S2). Four RT-PCR products contained either a 3′- mutant L1 expression plasmids (Fig. 1 C, lanes 2 to 9 and D), but truncated U6 snRNA cDNA sequence conjoined to a 5′- not in independent control experiments using untransfected HeLa truncated L1, a U6 snRNA followed by a partial hepatitis delta cells (Fig. 1C, lane 1). The cDNA products typically consisted of virus ribozyme (HDVr) sequence conjoined to a 5′-truncated L1, the 3′ end of U6 snRNA cDNA ending in ∼4to6thymidines and/or untemplated nucleotides at the U6/L1 junction. These conjoined to a variably 5′-truncated L1 sequence derived from the products also sometimes exhibited a microhomology of 1 to transfected L1 expression plasmid and closely resembled those of 3 nucleotides at the U6/L1 junction and structurally resemble previously characterized genomic U6/L1 pseudogenes (53, 55–58). previously described artifacts generated during RT-PCR (64) The constellation of U6/L1 cDNA products generally varied in (Fig. 2D and SI Appendix, Fig. S2C). size between ∼100 and ∼1,000 base pairs, depending upon where the 3′ end of U6 was conjoined to the 5′-truncated L1 sequence. U6/L1 RNA Ligation Requires a 2′,3′-Cyclic Phosphate and a 5′-OH. To The size and number of cDNA fragments for each transfection test whether the U6 2′,3′-cyclic phosphate was required for condition also varied between independent RT-PCR experiments. RtcB-mediated U6/L1 RNA ligation, purified RtcB was incu- Additional analyses revealed that there were not specific se- bated with a synthetic U6 snRNA that contained a 3′-OH ter- quences within L1 that facilitated U6/L1 chimeric RNA formation minus (U6-OH) and an L1 that contained a 5′ hydroxyl end (Fig. 1D and SI Appendix,Fig.S1B and Table S1). (OH-L1) (Fig. 2 A and B). In contrast to reactions with U6 > P, BLAT searches (63) revealed that the U6/L1 junction sequences the 305-bp cDNA diagnostic for bona fide U6/L1 chimeric cDNAs were not present in the human genome reference sequence (HGR/ was not overtly visible in reactions containing the U6-OH substrate build Grch38), indicating that they did not arise from the tran- (Fig. 2C, lane 4). The majority (39 of 43) of recovered cDNA scription of existing genomic U6/L1 pseudogenes. Notably, atypi- products appeared to be RT-PCR artifacts similar to those described cal U6/L1 chimeric cDNAs consisting of 3′-truncated U6 conjoined in the preceding paragraph (Fig. 2D and SI Appendix,Fig.S2C).

2of11 | www.pnas.org/cgi/doi/10.1073/pnas.1805404116 Moldovan et al. Downloaded by guest on September 24, 2021 pJM101/L1.3Δneo ORF1 ORF2 CMV 5’UTR EN RT pA A 3’UTR

pJM108/L1.3Δneo pJM105/L1.3Δneo pJBM119/L1.3Δneo

U6s1 SV40as cDNA synthesis Transfect Extract Total RNA oligo dT U6s2 3UTRas3 B U6 T pA 0 hrs ~48 hrs n 3’UTR Nested PCR

C ~bp 1) HeLa2) UTFpJM101/L1.3Δneo3) pJM101/L1.3Δneo4) pJM105/L1.3Δneo5) pJM105/L1.3Δneo6) pJBM119/L1.3Δneo7) pJBM119/L1.3Δneo8) pJM108/L1.3Δneo9) pJM108/L1.3Δneo10) H2011) H20 2000

1000

300 GENETICS 4387 pJM101/L1.3Δneo D 5’UTR ORF1 EN RT pJM101/ L1.3Δneo

U6 Tn L1.3Δneo pJM108/ pJM105/ L1.3Δneo pJBM119/ L1.3Δneo

Fig. 1. Chimeric U6/L1 RNA is generated in HeLa cells transfected with human L1 expression constructs. (A) Schematics of wild-type and mutant L1s. Gray rectangles represent 5′ UTR, inter-ORF space, and 3′ UTR, respectively; yellow rectangle, ORF1; blue rectangle, ORF2. L1s were cloned into the pCEP4 mammalian expression vector. A cytomegalovirus immediate early promoter (CMV, black rectangle) that augments L1 expression and an SV40 poly- adenylation signal (light blue rectangle, pA) flank the L1. The pJM101/L1.3Δneo plasmid expresses an active human L1 (L1.3). The pJM108/L1.3Δneo, pJM105/ L1.3Δneo, and pJBM119/L1.3Δneo plasmids express versions of L1.3 that contain mutations that render them unable to retrotranspose; the approximate locations of the respective mutations are indicated in the schematic. (B) Rationale of the RT-PCR experiments used to detect U6/L1 chimeric RNAs. HeLa cells were transfected with L1 expression plasmids, total cellular RNA was extracted ∼48 h posttransfection, and cDNAs were synthesized using an oligo-dT primer. Nested PCR was carried out using primers complementary to sequences within U6 and the 3′ end of the L1 construct (U6s1 and SV40as, then U6s2 and 3UTRas3). (C) Results from a representative RT-PCR experiment. The transfected L1 construct is indicated above each lane of the agarose gel image. Each lane contains a single biological replicate. Lane 1, HeLa UTF (untransfected HeLa cells); lanes 2 and 3, HeLa transfected with pJM101/L1.3Δneo; lanes 4 and 5, HeLa transfected with pJM105/L1.3Δneo; lanes 6 and 7, HeLa transfected with pJBM119/L1.3Δneo; lanes 8 and 9, HeLa transfected with pJM108/L1.3Δneo; lanes

10 and 11, H2O PCR controls. Molecular weight standards (in bp) are shown in the first and last gel lanes. At least 3 independent biological replicates were conducted for each transfection condition. (D) Structures of 38 U6/L1 chimeric RNAs found in transfected HeLa cells. U6/L1 RNA chimera sequences contain the

3′ terminus of U6 snRNA cDNA (white arrow) ending in ∼4 to 6 thymidine nucleotides (Tn) conjoined to a variable 5′−truncated L1. A schematic of the full- length L1.3 sequence is represented at the top of the schematic. The horizontal black lines indicate the approximate length of L1 sequence conjoined tothe U6 poly(T) tract. The 5′-most U6/L1 junction occurred at L1.3 nucleotide position 4387.

Moldovan et al. PNAS Latest Articles | 3of11 Downloaded by guest on September 24, 2021 A B U6 RNA L1 RNA 5’...ucgugaagcguuccauauuuu>P OH-cucaguaaacuaucgcaagaac...3’ |||||||||||||||||| |||||||||||||||||| 3’agcacttcgcaaggtata------catttgatagcgttcttg 5’ U6>P U6 UUUU>P DNA splint U6-OH U6 UUUU-3’OH RtcB Dnase I OH-L1 5’ OH L1 (5752-6087)

P-L1 5’PPP- L1 (5752-6087) U6s1 SV40as U6s2 3UTRas3

L1 U6 T4

No RT

C H2O + - - - - + - - - - OH-L1 + + + + - + + + + - P-L1 - - - - + - - - - + U6>P + + + - + + + + - + U6-OH - - - + - - - - + - RtcB - - + + + - - + + + O O 2 2 H RtcB* - + - - - - + - - - H 123456789101112 ~bp

400 305

200

100

Full-length 5’-truncated U6 T L1 U6 T L1 D Total 4 4 artifact U6>P/OH-L1 46 39 3 4 U6-OH/OH-L1 43 4 0 39 U6>P/P-L1 26 0 5 21

Fig. 2. Purified recombinant RtcB ligates U6 RNA to L1 RNA in vitro. (A) The rationale of the U6/L1 in vitro ligation experiment. A synthetic human U6 RNA containing a 2′,3′-cyclic phosphate (>P, red circle) and a synthetic L1 RNA (blue font) containing a 5′-OH (black circle) were generated using a ribozyme-based in vitro transcription reaction. U6 and L1 RNAs were splinted with a cDNA oligonucleotide (DNA splint, italics), and then the resultant RNA/DNA hybrid was incubated with purified RtcB (black rectangle, white font) for 1 h. The reaction was then treated with DNase I, the RNA was purified, and cDNAs were synthesized using the SV40as oligonucleotide primer. RT-PCR reactions using nested primers (U6s1 and SV40as, then U6s2 and 3UTRas3) were used to detect U6/ L1 chimeric cDNAs. (B) Schematic representations of the synthetic RNAs used in in vitro experiments. The in vitro transcribed U6 RNA ends in 4 uridine ribonucleotides and contains a 2′,3′-cyclic phosphate (U6 > P) or a 3′-OH (U6-OH). The in vitro transcribed L1 RNA consists of pJM101/L1.3Δneo sequence (nt positions 5752 to 6087) and contains a 5′-OH (OH-L1) or a 5′-triphosphate (P-L1). (C) Results from the in vitro U6/L1 ligation reactions. The constituents of U6/L1 ligation reactions are indicated above each gel lane (+) of the agarose gel image. An asterisk (*) indicates that RtcB was heat treated at 95 °C for 10 min prior to adding it to the reaction. No RT,

no RT control; H2O, water PCR controls. DNA size markers (in bp) are shown to the left of the gel image. The predicted position of the 305-bp U6/L1 RT-PCR product is noted on the left side of the gel image (white arrow, red font). (D) Summary of results from product characterization experiments. Column 1, synthetic RNAs used in the reaction; column 2, number of RT-PCR products characterized for each reaction condition; column 3, number of RT-PCR products that correspond to the full-length ligation product; column 4, number of RT-PCR products that contain a variably 5′-truncated L1 sequence; column 5, number of putative RT-PCR artifact products. Each in vitro experiment was repeated 3 independent times and yielded similar results.

Only 4 RT-PCR products contained a copy of the 3′ end of a U6 snRNA ending with a 2′,3′-cyclic phosphate and an L1 RNA that snRNA cDNA ending in 4 thymidine nucleotides precisely con- contains a 5′-OH end. joined to the 5′-OH of the full-length L1 RNA (Fig. 2D). To test whether an L1 5′-OH terminus was required for RtcB- U6 snRNA and L1 RNA Are Ligated in HeLa Cell Nuclear Extracts. We mediated U6/L1 RNA ligation, we incubated purified RtcB with next tested whether U6 and L1 RNA could be ligated in cell free U6 > P and an L1 RNA fragment containing a 5′-triphosphate extracts. Briefly, we incubated U6 > P and OH-L1 RNA in HeLa (P-L1) (Fig. 2 A and B). The 305-bp cDNA diagnostic for bona cell nuclear extracts and then used the same nested RT-PCR fide U6/L1 chimeras was not overtly visible in reactions that strategy used in the in vitro assay to detect U6/L1 chimeric contained P-L1 substrate (Fig. 2C, lane 5). We did not recover cDNAs (Fig. 2 A and B and Methods). Of note, we did not use a any full-length U6/L1 chimeric cDNAs (0 of 26) from these re- DNA oligonucleotide splint in these assays as endogenous RNase actions; however, 5 of 26 (∼19%) products contained the 3′ end H activity may result in the degradation of the complementary of a U6 snRNA cDNA ending in 4 thymidine nucleotides con- regions of the U6 > P and OH-L1 RNAs. Control Western blots joined to a 5′-truncated P-L1 (Fig. 2D and SI Appendix, Table confirmed the presence of RtcB in HeLa nuclear extracts (Fig. S2). As above, these products could result from the ligation of 3D and SI Appendix, Fig. S3A). U6 > P to broken or degraded L1 RNAs containing a 5′-OH. Similar to experiments using purified RtcB, the predicted 305-bp Thus, efficient RtcB-mediated ligation in vitro requires a U6 U6/L1 cDNA product was detected in reactions containing U6 > P

4of11 | www.pnas.org/cgi/doi/10.1073/pnas.1805404116 Moldovan et al. Downloaded by guest on September 24, 2021 and OH-L1 (Fig. 3A, lane 3) but was not visible in negative control essential gene (68) and the total loss of RtcB protein expression reactions that either lack or contain heat-treated HeLa cell nu- would prevent cell growth and/or viability (69, 70). clear extracts (Fig. 3A, lanes 1 and 2). Sequencing of RT-PCR To examine U6/L1 RNA ligation efficiency in RtcB-depleted products revealed that 47 of 106 (∼44%) products consisted of the HeLa nuclear extracts, we incubated the extracts with synthetic 3′ end of a copy of a U6 snRNA cDNA ending in 4 thymidine U6 RNA ending in a 2′,3′-cyclic phosphate and an L1 RNA that nucleotides conjoined precisely to the 5′-OH of the full-length contained a 5′ hydroxyl. Quantitative reverse transcriptase PCR OH-L1 RNA (Fig. 3B). By comparison, 38 of 106 (∼36%) prod- (RT-qPCR) revealed that U6/L1 ligation efficiency was reduced ucts consisted of the 3′ end of a copy of a U6 snRNA cDNA ∼4- to 5-fold in the RtcB2.1 and RtcB2.2 clonal cell lines, but that ending in 4 thymidine nucleotides conjoined to a 5′-truncated neither RtcB protein levels nor U6/L1 ligation efficiency was af- OH-L1 (Fig. 3B). The prevalence of U6/L1 chimeras conjoined fected in a clonal cell line containing a negative control sgRNA to 5′-truncated L1 sequences was higher in reactions conducted that targeted GFP (Fig. 3 D and E). with HeLa cell nuclear extracts when compared to in vitro reac- To determine whether ectopic RtcB expression could com- 2.1 2.2 tions conducted with purified RtcB (36% vs. 7%), suggesting that plement the defect in the RtcB and RtcB cell lines, we L1 RNA may be cleaved by a ribonuclease activity that generates transfected them with an RtcB cDNA expression plasmid. Western 5′-OH ends in the HeLa cell nuclear extracts. As described above, blots revealed that ectopic RtcB expression led to an ∼1.4-fold and 2.1 we also recovered 21 of 106 (∼20%) putative artifactual RT-PCR ∼1.5-fold increase in RtcB protein expression in the RtcB and 2.2 products (64) (SI Appendix,Fig.S3B). RtcB clonal cell lines when compared to HeLa-JVM cells (Fig. Incubation of the U6-OH and OH-L1 substrates in HeLa cell 3D). RT-qPCR experiments revealed that ectopic RtcB expression ∼ 2.1 nuclear extracts did not yield the predicted 305-bp U6/L1 cDNA partially rescued ligation efficiency by 2-fold in the RtcB and 2.2 E product (Fig. 3A, lane 4). DNA sequence analyses revealed that RtcB extracts (Fig. 3 ). These data indicate that endogenous 38 of 43 (∼88%) products likely represent artifacts generated RtcB contributes to U6/L1 RNA ligation in HeLa cell extracts. during RT-PCR (64) (SI Appendix,Fig.S3B). Four out of 38 (∼9%) products contained the 3′ end of a U6 snRNA cDNA U6 snRNA and GFP Are Ligated in HeLa Cell Nuclear Extracts. The ending in 5 thymidine nucleotides conjoined precisely to the 5′- HGR contains chimeric pseudogenes that consist of a full-length OH of the full-length OH-L1 RNA substrate. The synthetic U6 > copy of U6 conjoined to cDNAs derived from other cellular mRNAs (58), suggesting that U6 RNA can be ligated to non-L1 RNAs. To P RNA ends in 4 uridine ribonucleotides whereas the mature

test this hypothesis, we generated a 310-nucleotide RNA fragment GENETICS forms of human U6 snRNA typically end in 5 uridine ribonucle- that corresponds to the 3′-end of a humanized Renilla GFP cDNA otides (45); thus, endogenous U6 snRNA may have been ligated that contains a 5′-OH (OH-GFP) (Methods). We incubated U6 > P to the OH-L1 RNA in these reactions. Finally, 1 product con- and OH-GFP RNA in HeLa cell nuclear extracts and then used RT- tained the 3′ end of a copy of a U6 snRNA cDNA ending in PCR to detect cDNA products (SI Appendix,Fig.S3E and F and 4 thymidine nucleotides followed by 5′-truncated L1 (Fig. 3B). Methods). Incubation of the U6 > P and OH-GFP RNA substrates Thus, a U6 2′,3′-cyclic phosphate is required for efficient U6/L1 with HeLa cell nuclear extracts yielded the predicted 232-bp cDNA ligation in HeLa cell nuclear extracts. SI Appendix G ′ product ( ,Fig.S3 ,lane3). Sequencing of the cDNA To test whether a 5 -OH was required for U6/L1 ligation, we in- products revealed that 9 of 23 (39%) consisted of a copy of the 3′ cubated U6 > P and P-L1 RNAs in HeLa cell nuclear extracts. We A end of a U6 snRNA ending in 4 thymidine nucleotides conjoined did not detect the predicted 305-bp U6/L1 cDNA product (Fig. 3 , precisely to the 5′-OH of the full-length GFP RNA fragment lane 5). Sequence analyses did not reveal the presence of the pre- whereas 6 of 23 (26%) contained a copy of the 3′ end of a dicted U6/L1 chimeric cDNA; however, 16 of 23 (∼70%) of the ′ ′ U6 snRNA ending in 4 thymidine nucleotides followed by a 5 - cDNA products consisted of a copy of the 3 end of a U6 snRNA truncated GFP RNA fragment (SI Appendix,Fig.S3G and H). cDNA ending in 4 thymidine nucleotides followed by a variably 5′- ∼ B The remaining 8 of 23 ( 35%) cDNA products contained either a truncated P-L1 sequence (Fig. 3 ). These U6/L1 junctions occurred 3′-truncated U6 snRNA sequence conjoined to a 5′-truncated GFP, throughout L1 RNA, and there was not an overt sequence motif U6 snRNA followed by a partial HDVr sequence conjoined to a 5′- C SI within L1 RNA that facilitated U6/L1 formation (Fig. 3 and truncated GFP, and/or untemplated nucleotides at the U6/GFP Appendix C ,Fig.S3 and Table S3).Thesedataareconsistentwith junction. These sequences sometimes exhibited 1 to 3 nucleotide > results from reactions where U6 P and OH-L1 were incubated microhomologies at the U6/L1 junction and likely represent arti- with HeLa cell nuclear extracts, suggesting that L1 RNA may be facts generated during RT-PCR (similar in structure to SI Appendix, processed by a ribonuclease activity in the HeLa cell nuclear ex- Fig. S2C, except GFP replaces L1) (64). These data suggest that tracts either prior to or during the ligation reaction. The remaining chimeric RNA formation is not exclusive to L1 RNA. 7of23(∼30%) cDNA products were characterized as putative RT-PCR artifacts (64) (SI Appendix,Fig.S3B). Thus, efficient U6/ Endogenous U6/L1 RNA Is Part of the Transcriptome in Human Cells. L1 ligation in HeLa cell nuclear extracts requires U6 2′,3′-cyclic Data from transfection-based experiments and in vitro ligation phosphate and 5′-OH L1 substrates. experiments suggested that U6 snRNA could be ligated to L1 RNA in vivo; thus, we sought to determine whether U6/L1 chi- Depletion of RtcB from HeLa Cells Affects U6/L1 Ligation Efficiency. meric RNAs were part of the transcriptome in human cells. We Ligation reactions using HeLa nuclear extracts suggested that searched for U6/L1 junction reads in 100-base pair paired-end RtcB might ligate U6/L1 RNA in HeLa cells. To test this hy- RNA sequencing (RNA-seq) data generated from 2 independent pothesis, we utilized CRISPR/Cas9 gene editing to generate 2 HeLa cell lines (HeLa-JVM and HeLa-HA), a human embryonic 2.1 2.2 HeLa cell lines (RtcB and RtcB ) exhibiting reduced RtcB carcinoma cell line (PA-1), a human embryonic stem cell line protein expression (SI Appendix, Methods). Sanger sequencing (H9-hESCs), and H9-derived neural progenitor cells (NPCs) revealed that the RtcB alleles in RtcB2.1 and RtcB2.2 contain (Fig. 4A and SI Appendix, Methods). Each of these cell lines can genomic edits that are predicted to result in either frame-shift accommodate the retrotransposition of engineered human L1s mutations or in-frame deletions in the RtcB amino acid sequence (22, 71–74). We identified 398 U6/L1 chimeric RNA read-pairs (SI Appendix, Fig. S3D). Western blots confirmed that steady out of ∼1.1 × 109 RNA sequencing reads across the 5 cell lines. state RtcB protein levels were reduced by ∼80% and ∼73% for After removing duplicate PCR reads, we then merged overlapping the RtcB2.1 and RtcB2.2 clonal cell lines, respectively (Fig. 3D). reads to identify 64 intact U6/L1 junction sequences. We were unable to isolate a HeLa cell clone with a complete Hand annotation of the 64 U6/L1 junctions revealed that 53 knockout of RtcB protein expression, likely because RtcB is an (∼83%) consisted of the 3′ end of U6 snRNA cDNA ending in

Moldovan et al. PNAS Latest Articles | 5of11 Downloaded by guest on September 24, 2021 ACNo RT 5752 6087 H2O + - - - - + - - - - OH-L1 + + + + - + + + + - L1 P-L1 - - - - + - - - - + U6>P + + + - + + + + - + U6-OH - - - + - - - - + - Extract - - + + + - - + + + O O 2 2 H Extract* - + - - - - + - - - H 1 2 3 4 5 6 7 8 9 10 11 12 ~bp 400 305 200

100

Full-length 5’-truncated U6 T L1 U6 T L1 B Total 4 4 artifact U6>P/OH-L1 106 47 38 21 U6-OH/OH-L1 43 4 1 38 U6>P/P-L1 23 0 16 7

123456789101112 D RtcB2.1 RtcB2.1 RtcB2.2 RtcB2.2 ~kDa 2.1 2.1 2.2 2.2 HeLa HeLa GFPGFP RtcB RtcB RtcB RtcB RescueRescueRescue Rescue

100 NCL 75 RtcB 50

n.s. 2.0 1.5 1.38

1.0 1.00 0.5 0.65 0.38 0 0.20 0.26 RtcB2.1 RtcB2.2 2.1 2.2 HeLa GFP RtcB Rescue RtcB Rescue

Fig. 3. HeLa cell nuclear extracts mediate the ligation of U6 and L1 RNAs. (A) Results from U6/L1 ligation reactions using HeLa cell nuclear extracts. The ligated U6/ L1 RNA was purified from ligation reactions and analyzed using RT-PCR and agarose gel electrophoresis. The constituents of U6/L1 ligation reactions are indicated aboveeachlane(+) of the representative agarose gel image. An asterisk (*) indicates that the HeLa cell nuclear extract was heat treated at 95 °C for 10 min prior to

adding it to the reaction. No RT, no RT control; H2O, water PCR controls. DNA size markers (in bp) are shown to the left of the gel image. The predicted position of the 305-bp U6/L1 RT-PCR product is noted on the left side of the gel image (white arrow, red font). (B) Summary of results of ligation reactions using HeLa cell extracts. Column 1, RNAs used in the reaction; column 2, number of RT-PCR products characterized for each reaction condition; column 3, number of RT-PCR products that correspond to the full-length ligation product; column 4, number of RT-PCR products that contain a variably 5′-truncated L1 sequence; column 5, number of putative RT-PCR artifact products. (C) Structures of U6/L1 chimeric RNAs containing 5′-variably truncated L1 sequences. A schematic of the L1 fragment used as a template for the in vitro transcription reaction is represented at the top. The horizontal black lines indicate the approximate length of the L1 sequence conjoined to the U6 poly(T) tract. U6/L1 chimeric RNAs were isolated from 19 independent experiments. (D) Depletion of RtcB protein expression in HeLa cell nuclear extracts. Western blot images depicting RtcB expression (green arrow) in HeLa nuclear extracts. Extract sources are indicated above each lane (HeLa indicates untransfected HeLa extracts). Each lane represents an independent biological replicate. The Western blot experiment was done twice. Nucleolin (NCL) (red arrow) was used as a loading control. Approximate molecular weights are indicated to the left of the gel image. (E) Depletion of RtcB affects U6/L1 ligation efficiency in HeLa extracts. The x axis indicates the experimental condition. The y axis indicates the normalized U6/L1 ligation efficiency. Ligation efficiencies were normalized to untransfected HeLa-JVM extracts, which are set to 1. The ligation efficiency value represents the average of 6 independent RT-qPCR experiments. Error bars indicate SDs. Two-tailed t tests were used to determine significance. An asterisk (*) indicates P value < 0.05; n.s., not significant.

∼4 to 8 thymidine nucleotides conjoined to a variably 5′-truncated U6/L1 chimera formation (Fig. 4B and SI Appendix,Fig.S4A and L1 sequence (Fig. 4B and SI Appendix, Tables S4–S6). Of note, B). The remaining 11 of 64 (∼17%) U6/L1 sequences contained a 4 of these 53 U6/L1 chimeras consisted of U6 ending in 5 to 3′-truncated U6 snRNA conjoined to a 5′-truncated L1 and were 7 thymidines conjoined to an L1 sequence in an antisense orien- excluded from further analysis as they were structurally similar tation, suggesting that U6 can become conjoined to both sense to template-switching artifacts generated during cDNA synthesis and antisense L1 RNAs (SI Appendix,TableS6). As above, there described above (64) (SI Appendix, Table S6). Thus, 53 bona fide was not a specific sequence within L1 that appeared to facilitate unique U6/L1 chimeras were subjected to further analysis.

6of11 | www.pnas.org/cgi/doi/10.1073/pnas.1805404116 Moldovan et al. Downloaded by guest on September 24, 2021 A 1)RNA fragmented to ~190 nucleotides and subjected to 100 bp paired end seq.

2) Align reads to masked genome reference U6 L1 U6 L1 spiked with 1 copy each of U6 and L1 Masked HGR

3)Assemble U6/L1 read pairs U6 TTTT L1L1 U6 TTTT L1L1 no overlap overlapping

4)Align U6/L1 junctions to unmasked genome U6 TTTT L1L1 aligned non-aligned B L1.3 5’UTR ORF1 EN RT HeLa JVM

HeLa HA

NPC

PA-1

aligned Replicate 1

Replicate 2 GENETICS

U6 Tn

non-aligned

Fig. 4. RNA-seq detection of endogenous U6/L1 chimeric RNAs in human cell lines. (A) Rationale of the RNA-seq experiments. Step 1: Ribosome-depleted RNA was fragmented to ∼190 nucleotides and subjected to 100-bp paired-end DNA sequencing. Step 2: RNA-seq read pairs (arrows) were aligned to a repeat masked version human reference genome (HGR/build Grch38), which contained “spiked-in” copies of a single U6 (white rectangle) and single L1.3 (blue rectangle) sequence. RNA-seq reads that did not map to U6 or L1 were discarded from subsequent analyses. Step 3: Overlapping U6/L1 read pairs were merged to determine the U6/L1 junction sequences. U6/L1 read pairs that contained a gap (i.e., “no overlap”) were discarded from subsequent analyses. Step 4: Overlapping U6/L1 junctions were aligned to the unmasked HGR. If the U6/L1 junction mapped to the HGR with >90% accuracy, it was designated as an “aligned” read. U6/L1 junctions that failed to map to the HGR with at least 90% accuracy were designated as “non-aligned” reads. (B) Structures of RNA-seq U6/L1 junctions. A schematic of a full-length RC-L1 is indicated at the top. The general structure of a U6/L1 chimeric junction sequence consists of the 3′ end of a U6 snRNA

cDNA sequence ending in ∼4 to 8 thymidine nucleotides (left side of figure; white arrow ending in Tn) conjoined to a variably 5′−truncated L1 sequence. Two independent RNA-seq libraries were generated from HeLa-JVM, H9, NPC, and PA-1 cells, respectively (squares and triangles, respectively). One RNA-seq library was generated from HeLa-HA cells. Red horizontal lines, HeLa-JVM; green horizontal lines, HeLa-HA; yellow horizontal lines, PA-1; black horizontal lines, H9; blue horizontal lines, human NPCs. Each horizontal dashed line represents a single U6/L1 junction RNA-seq merged sequence read. The triangle or square at the left end of the horizontal dashed lines indicates the approximate location of the U6/L1 junction point relative to L1.3. The top set of dashed lines represent 16 U6/L1 junction sequences that mapped (“aligned”) to the HGR. The bottom set of dashed lines represent 33 U6/L1 junction sequences that did not map (“non- aligned”) to the HGR. These 33 U6/L1 junctions contained a copy of U6 conjoined to an L1 present in the same transcriptional orientation. The remaining 4 U6/L1 chimeras that did not map to the HGR (SI Appendix,TableS6) contained a copy of U6 conjoined to an L1 present in the opposite transcriptional orientation.

Most U6/L1 Chimeric RNA Sequences Do Not Align to the Genome. in RNA-seq experiments were derived from the transcription of The low proportion of U6/L1 chimera RNAs in our dataset an existing genomic U6/L1 or represented unique chimeric suggested that they might represent a rare subset of RNAs in RNAs, the 53 U6/L1 chimeric RNAs were used as probes in human cells. To determine whether the U6/L1 chimeras detected BLAT searches of the HGR (Fig. 4A and SI Appendix, Methods).

Moldovan et al. PNAS Latest Articles | 7of11 Downloaded by guest on September 24, 2021 Sixteen out of 53 (∼30%) U6/L1 junctions were present in the mechanistic explanation for how RtcB can mediate the ligation of HGR, suggesting that they could have resulted from the tran- U6 snRNA to a diverse cohort of cellular RNAs containing 5′-OH scription of extant U6/L1 pseudogenes. Seven out of the 16 pu- ends. It remains possible that another RNA ligation activity pre- tative transcribed U6/L1 chimeric RNAs were detected in sent in HeLa cells could also contribute to U6/L1 chimera for- multiple cell lines (Fig. 4B and SI Appendix, Table S6), and 7 mation. For example, previous studies have shown that a yeast-like were supported by multiple reads from the same cell line (Fig. 4B tRNA ligase activity can join 2′,3′-cyclic phosphate and 5′-OH and SI Appendix, Table S6). The 16 genomic U6/L1 chimeric containing RNAs in vertebrate cells; however, this ligase remains pseudogenes that served as putative transcription templates that to be identified (80). gave rise to chimeric U6/L1 RNAs exhibited L1 structural hallmarks (SI Appendix, Table S7). They consisted of a full-length RNA Ligation in HeLa Cell Extracts. U6/L1 ligation reactions using U6 snRNA sequence ending in 5 to 7 thymidine nucleotides purified bacterial RtcB required an oligonucleotide splint, pre- conjoined to a variably 5′-truncated L1, were flanked by 6- to 19-bp sumablytokeeptheU6snRNA2′,3′-cyclic phosphate and L1 5′-OH target site duplications, and inserted into an L1 EN consensus ends in close proximity to one another. In contrast, U6/L1 ligation cleavage sequence. By comparison, 37 out of 53 (∼70%) U6/L1 reactions using HeLa cell nuclear extracts did not require an oli- junction sequences did not align to the HGR and were unique to a gonucleotide splint. The human form of RtcB is 505 amino single cell line (Fig. 4B). Thirty-one of 37 junctions were sup- acids in length, and protein sequence alignments using the ported by a single merged read pair, 5 of 37 junctions were Universal Protein Resource (UniProt) align tool (81) showed supported by 2 merged reads, and 1 junction was supported by that human RtcB is highly conserved (>90%) among other 3mergedreads(Fig.4B and SI Appendix, Table S6 and Methods). vertebrate forms of RtcB (SI Appendix, Fig. S3I). However, hu- Human-specific L1 insertions can be polymorphic with respect man RtcB only shares ∼25% amino acid identity with RtcB from to presence/absence in the human population (75); thus, it is Escherichia coli (408 amino acids in length) (SI Appendix, Fig. conceivable that some of the cell lines used to generate RNA- S3I), suggesting that changes to the RtcB amino acid sequence seq data could contain a genomic U6/L1 chimeric pseudogene over evolutionary time could have affected RtcB enzymatic that is absent from the HGR. To examine this possibility, we activity and/or function. used the 53 U6/L1 junctions as probes to query HeLa genome It is also possible that cellular proteins within HeLa nuclear sequencing data available in the database of Genotypes and extracts may help facilitate U6/L1 RNA ligation. Previous stud- Phenotypes (dbGaP accession number phs000640.v1.p1) (76– ies have revealed that L1 RNPs are associated with numerous 78). Controls revealed that the 16 U6/L1 junction sequences that RNA binding proteins and cellular RNAs, including U6 snRNA aligned to the HGR were also present in the HeLa genome data (82–87). Moreover, RtcB has been detected in GFP-tagged L1 (Fig. 4B and see Methods). By comparison, the 37 non-aligned ORF1p cytoplasmic granules (83), cytoplasmic stress granules U6/L1 junction sequences were absent from HeLa genome (88), and other protein/RNA complexes within the cell (60, 89, data. 90) whereas U6 snRNA associates with cellular RNA binding To further validate the uniqueness of the 37 U6/L1 junction proteins, including La and the Lsm2-8 ring (38, 91, 92). Of note, sequences, we aligned the 53 U6/L1 junction sequences to 23 the Lsm2-8 ring is ejected from U6 during splicing, and U6 high-coverage individual genomes representing 23 distinct hu- snRNA must be recycled, which could provide other cellular man geographic populations from the 1000 Genomes Project proteins access to the 3′ end of U6 snRNA. Thus, it is plausible dataset (SI Appendix, Table S8 and Methods) (79). The 16 U6/L1 that protein–protein or protein–RNA interactions within HeLa junction sequences that were present in the HGR and HeLa cell cell nuclear extracts may help bring U6 snRNA and L1 RNA genomic datasets also were present in each of the 23 of high into close proximity to facilitate ligation. coverage 1000 Genomes Project individual genomes; 2 genomes (NA20845 and HG03742) contained an SNP in the U6 portion The L1 RNA May Be Processed by Endoribonuclease Activity in HeLa of the junction sequences (SI Appendix, Table S6). In contrast, Cells. In contrast to experiments conducted with recombinant the 37 non-aligned U6/L1 junction sequences were absent from bacterial RtcB, almost half of the U6/L1 ligation products the high coverage 1000 Genomes Project individual genomes. characterized using HeLa cell nuclear extracts consisted of the 3′ Thus, these data suggest that the 37 U6/L1 junctions detected in end of U6 snRNA cDNA ending in 4 thymidine nucleotides our RNA-seq experiments do not correspond to existing U6/L1 conjoined to a 5′-truncated L1 (Fig. 3 A and B and SI Appendix, genomic pseudogenes and that different cell types may contain a Table S3). This result was evident in reactions with L1 RNA unique cohort of chimeric RNAs that are generated by post- substrates containing either a 5′-OH or a 5′ triphosphate. Given transcriptional RNA ligation events. that efficient ligation reactions were dependent upon U6 snRNA ending in a 2′,3′-cyclic phosphate and L1 RNA with a 5′-OH, Discussion these data raise the possibility that L1 RNAs may be processed RNA Ligation Generates Chimeric U6/L1 RNA. Here, we demon- by an endogenous endoribonucleolytic activity within HeLa cell strated that U6/L1 chimeric RNAs are generated in transfected nuclear extracts prior to their ligation to U6 snRNA. A number HeLa cells independently of L1 retrotransposition (Fig. 1 C and of cellular endoribonucleases generate RNA fragments with 5′- D). In vitro ligation assays demonstrated that purified RtcB can OH ends (93), including the tRNA splicing endonuclease (66), ligate U6 snRNA substrates ending in a 2′,3′-cyclic phosphate to RNase L (94, 95), angiogenin (96, 97), and inositol-requiring L1 RNAs containing a 5′-OH (Fig. 2 C and D) and that re- enzyme 1 (IRE1) (98). RNase L, for example, can target viral as placement of either the U6 2′,3′-cyclic phosphate with a 3′-OH or well as host cellular RNAs (94, 95, 99) and inhibits L1 retro- the L1 5′-OH with a 5′ triphosphate dramatically reduced the transposition by targeting L1 RNA (100). Recent reports have formation of RtcB-mediated ligation products. Additional assays also provided evidence that RtcB associates with IRE1, an revealed that HeLa cell nuclear extracts can mediate ligation of a endoribonuclease involved in the UPR (69, 98). It is possible that U6 snRNA substrate ending in a 2′,3′-cyclic phosphate to an L1 other classes of RNA endoribonucleases and/or exoribonucleases RNA containing a 5′-OH (Fig. 3A), depleting RtcB from HeLa that generate RNA 5′-phosphate ends also could process the L1 extracts decreases U6/L1 ligation efficiency (Fig. 3E), and U6 RNA. However, in these instances, the 5′-phosphate end would RNA could be ligated to non-L1 RNAs (SI Appendix,Fig.S3G). need to be hydrolyzed by a 5′-phosphatase to generate a 5′-OH Finally, RNA-seq experiments revealed that endogenous U6/L1 end suitable for ligation by RtcB. Thus, the most parsimoni- RNA chimeras are a component of the transcriptome of multi- ous explanation of the data is that the L1 RNA is processed by ple human cell types (Fig. 4B). Together, these data provide a an as-yet-unknown endoribonucleolytic activity within HeLa cell

8of11 | www.pnas.org/cgi/doi/10.1073/pnas.1805404116 Moldovan et al. Downloaded by guest on September 24, 2021 nuclear extracts to generate a truncated L1 RNA containing a5′-OH.

Chimeric U6/L1 RNAs Are Present in Human Cells. RNA-seq experiments demonstrated that U6/L1 chimeric RNAs are a component of the transcriptome in human cancer cell lines, hESCs, and human NPCs (Fig. 4B). The majority (∼70%) of U6/L1 chimeric (1) (5)

RNA-seq reads failed to align to the HGR, HeLa, or 23 high- AAAAAAAAA AAAAAA 5’ 5’ coverage genomes in the 1000 Genomes Project dataset (Fig. L1 RNA 4B), suggesting that they were generated de novo by a posttran- scriptional mechanism that results in the formation of a unique (6) RtcB (4) U6/L1 chimeric RNA molecule. Consistent with this conclusion, “ ” AAAAAA the majority of the non-aligned junctions were supported by a 5’ OH 5’ >P single RNA-seq read and are unique to a single cell line. In U6 snRNA contrast, ∼30% of the U6/L1 chimeric RNAs aligned to the HGR, HeLa, and 23 high-coverage genomes in the 1000 Genomes Project dataset (Fig. 4B), indicating that they are generated (3) from existing U6/L1 pseudogenes. Of note, 13 of these U6/L1 pseudogenes are embedded within the introns of annotated AAAAAA SI Appendix 5’ RefSeq genes ( ,TableS7), with 8 oriented in the L1 RNP (2) same transcriptional orientation as the gene, suggesting that U6/L1 chimeric RNAs could be expressed as part of an RNA Fig. 5. A model of U6/L1 RNA pseudogene formation. Following tran- polymerase II pre-mRNA transcript. Vertebrate U6 snRNA is scription, L1 RNA (black wavy line) is exported to the cytoplasm (step 1). L1 transcribed by RNA polymerase III and relies on upstream RNA is translated, and the L1 proteins, ORF1p (yellow circles) and ORF2p promoter elements to drive its transcription (38, 40). Thus, unless (blue circle) bind to their encoding L1 RNA to form an L1 ribonucleoprotein U6/L1 chimeric pseudogenes fortuitously inserted downstream of a particle (RNP) (step 2). After RNP formation, the L1 RNA is cleaved by an unidentified endoribonuclease to generate 5′-truncated L1 RNA with a 5′-

promoter that could augment RNA polymerase III transcription, it OH (step 3). RtcB, or a related ligase, ligates U6 snRNA to 5′-truncated L1 GENETICS remains unlikely that U6/L1 chimeric RNAs are transcribed as RNA (step 4). The resultant chimeric U6/L1 RNA is inserted into genomic DNA discrete transcription units. by TPRT (in cis), which results in the formation of a U6/L1 chimeric pseudogene (white band on black chromosome) (step 5). It is possible that L1 RNA could A Model of U6/L1 Pseudogene Formation. U6 snRNA is enriched in also be processed by nuclease activity (red scissors) and ligated to U6 while still the nucleus (101) whereas RtcB is present in both the nucleus in the nucleus (step 6). In this scenario, it is possible the chimeric U6/L1 RNA and cytoplasm (69, 102). Our data, in conjunction with previous could undergo retrotransposition by trans-complementation. knowledge of the L1 retrotransposition cycle, lead us to propose a mechanism for how U6/L1 chimeric pseudogenes are generated in human cells (Fig. 5). We posit that a ribonucleoprotein ther of which may serve as a substrate for ligation by RtcB to a ′ complex that minimally contains U6 snRNA, RtcB, and an un- 5 -OH (66, 67). defined endoribonuclease can cleave the L1 RNA present in wild-type L1 RNPs to generate a 5′-OH end, which, in turn, al- Conclusions. We provide mechanistic evidence for how U6/L1 lows L1 RNA to be ligated to the U6 snRNA 2′,3′-cyclic phos- chimeric pseudogenes are formed in the human genome. Our phate. As L1 ORF2p preferentially associates with the L1 data suggest that, in addition to their roles in mRNA and tRNA poly(A) tail (23, 31), the resultant chimeric U6/L1 RNAs likely splicing, U6 snRNA and RtcB are involved in the formation of retrotranspose in cis. The majority of chimeric pseudogenes chimeric cellular RNAs, thereby contributing to RNA tran- identified in the HGR consist of the full-length U6 snRNA se- scriptome diversity. The presence of a 2′,3′-cyclic phosphate on quence ending in a poly(T) tract conjoined to a variably 5′- human U6 snRNA stabilizes it and prevents it from degradation truncated L1 sequence (53, 55, 57, 58); thus, this mechanism (38). We speculate that RtcB-mediated ligation of U6 to L1 or provides a plausible explanation for the generation of most U6/L1 other cellular RNAs containing a 5′-OH would eliminate the U6 chimeric pseudogenes. U6atac snRNA also ends in a 2′,3′-cyclic 2′,3′-cyclic phosphate, which could, in principle, facilitate U6 phosphate (103); thus, the above model also could explain how snRNA turnover. U6atac/L1 chimeric pseudogenes are generated in human cells (55, 57). Methods Our data further demonstrate that U6 snRNA can become Oligonucleotide Sequences. Complete methods on cell culture, RT-PCR, RNA conjoined to retrotransposition-defective L1 and other cellular ligation assays, CRISPR gene editing, quantitative real-time PCR, and RNA- RNAs. However, because the retrotransposition of these RNAs seq can be found in SI Appendix, Methods. A list of oligonucleotides used in would need to coopt the L1-encoded proteins in trans (31), they this study is provided in SI Appendix, Table S9. The University of Michigan probably occur less frequently than U6/L1 chimeric pseudogenes Pluripotent Stem Cell Research Oversight (HPSCRO) Committee has approved that are formed in cis. Our model does not rule out the possi- work with hESCs in the Moran lab (Record no. 1004/1023). bility that some snRNA/L1 chimeric pseudogenes arise during TPRT by a template-switching mechanism. For example, a ACKNOWLEDGMENTS. We thank Dr. Mitsuhiro Nakamura and Mr. Trenton Frisbie for critical reading of this manuscript and helpful discussions; and small number of retro-pseudogene sequences consist of other Dr. Diane Flasch and the University of Michigan DNA Sequencing Core Facility small RNA species (e.g., U1, U3, U5, and 5s rRNA), which tend for generating the RNA-seq datasets. Henrietta Lacks and the HeLa cell line to be 3′-truncatedandareconjoinedtoeither5′-truncated L1s that was established from her tumor cells without her knowledge or consent or other cellular RNAs (53, 55, 57, 58). As these small RNAs in 1951 have made significant contributions to scientific progress and advances are not normally modified at their 3′ ends to contain a 2′,3′-cyclic in human health. We are grateful to Henrietta Lacks, now deceased, and to phosphate, it remains possible that the resultant chimeric products her surviving family members for their contributions to biomedical research. ′ J.V.M. was supported by National Institutes of Health (NIH) Grants are formed by an RtcB-independent mechanism, or the 3 trun- GM060518 and U01MH106892 and funds from the Howard Hughes Medical cation of the small RNAs is created by an endoribonuclease that Institute. R.E.M. was supported by NIH Grant HG007068. S.S. was supported generates a 2′,3′-cyclic phosphate or 3′-monophosphate end, ei- by NIH Grant R35-GM126945.

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