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HSPC117 Is the Essential Subunit of a Human tRNA Splicing Ligase Complex

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Science  11 Feb 2011:
Vol. 331, Issue 6018, pp. 760-764
DOI: 10.1126/science.1197847

Abstract

Splicing of mammalian precursor transfer RNA (tRNA) molecules involves two enzymatic steps. First, intron removal by the tRNA splicing endonuclease generates separate 5′ and 3′ exons. In animals, the second step predominantly entails direct exon ligation by an elusive RNA ligase. Using activity-guided purification of tRNA ligase from HeLa cell extracts, we identified HSPC117, a member of the UPF0027 (RtcB) family, as the essential subunit of a tRNA ligase complex. RNA interference–mediated depletion of HSPC117 inhibited maturation of intron-containing pre-tRNA both in vitro and in living cells. The high sequence conservation of HSPC117/RtcB proteins is suggestive of RNA ligase roles of this protein family in various organisms.

Transfer RNAs (tRNAs) are essential adaptor molecules in the translation of the genetic transcript into proteins. During their posttranscriptional maturation (1), intron-containing tRNA precursor transcripts (pre-tRNAs) undergo splicing, which is accomplished by a specialized endonuclease that excises the intron (2, 3) and a ligase that joins the resulting exon halves (fig. S1A). Although introns within pre-tRNA transcripts have been detected in all domains of life, the splicing machinery diverged at the ligation step during evolution (4). In fungi and plants, multifunctional proteins (57) homologous to bacteriophage T4 RNA ligase 1 (T4 Rnl1) (8) prepare the exon termini by the action of cyclic phosphodiesterase and polynucleotide kinase activities before catalyzing the actual ligation (fig. S1A, upper branch) (9, 10). In contrast, the animal and archaeal clades ligate tRNA exons by directly joining the 2′,3′-cyclic phosphate (RNA>p) and 5′-OH termini left after cleavage, leading to incorporation of the precursor-derived 2′,3′-cyclic phosphate into the splice junction (fig. S1A, lower branch) (1114). No RNA>p ligase has been identified since the direct tRNA splicing pathway was initially postulated.

By serendipity, it was discovered that 3′-phosphorylated (3′-P), 5′-OH double-stranded RNA (dsRNA) molecules become covalently linked upon incubation with human cell extracts (Fig. 1A, upper panel, and fig. S1, B and C) (15, 16) after their conversion into 2′,3′-cyclic phosphate–terminated dsRNA (11, 17). Removal of the 3′-phosphate, phosphorylation of the 5′-OH, or a combination of both inhibited interstrand ligation (Fig. 1A, lower panel). Therefore, we decided to use 3′-P dsRNA as a stable surrogate substrate for human tRNA ligase. By monitoring interstrand ligation, we were able to follow RNA>p ligase activity along three chromatographic purification steps (fig. S1, D and E) and used the resulting RNA>p ligase–enriched MonoQ fraction to identify 91 proteins by tryptic digestion and tandem mass spectrometry (MS) (table S1). HSPC117/C22ORF28, a member of the uncharacterized protein family UPF0027 (18), appeared to be of particular interest, as it is the human homolog of the bacterial and archaeal RtcB gene, which, together with RtcA, the RNA 3′-P terminal cyclase, resides within a σ54-regulated operon in Escherichia coli (19). As a consequence, HSPC117/RtcB proteins have been predicted to function in RNA processing or modification (20). Both RNA>p ligase activity and the UPF0027 protein family are not detectable in plant and fungal model organisms (e.g., Arabidopsis thaliana or Saccharomyces cerevisiae) (4, 18), suggesting a link between HSPC117 and direct RNA ligation.

Fig. 1

Identification of the RNA>p ligase HSPC117. (A) (Upper panel) Scheme illustrating dsRNA interstrand ligation. 5′-OH, 3′-P RNA oligonucleotides (depicted in gray; asterisk marks position of radiolabel) were incubated with (lanes a and b) or without (lanes c and d) alkaline phosphatase and annealed to complementary RNA strands (depicted in black) incubated with (lanes a and c) or without (lanes b and d) T4 Pnkp and ATP. The obtained RNA duplexes were used as substrates for interstrand ligation in HeLa cell extracts (lower panel). (B and C) Dilution series of protein extracts prepared from cells transfected with small interfering RNAs (siRNAs) targeting HSPC117 and enhanced green fluorescent protein (EGFP) as a control were assayed for interstrand ligation (B) and tRNA maturation (C). (D) Depletion of HSPC117 was confirmed by Western blot.

Depleting HSPC117 by RNA interference (RNAi) abolished interstrand ligation (Fig. 1B and fig. S2) and impaired processing of pre-tRNA transcripts into mature tRNA in cell extracts, leading to a concomitant accumulation of exon halves. These findings are consistent with a role in the ligation of tRNA exons (Fig. 1, C and D).

Assuming that mutation of a strictly conserved cysteine residue (fig. S3) predicted to be involved in metal ion coordination in RtcB proteins (21, 22) would render the enzyme inactive, we created clonal cell lines expressing wild-type c-myc–HSPC117 and the point mutant C122A (Cys122 → Ala122). Affinity purification of c-myc–HSPC117 yielded an immunoprecipitate ligating tRNA exon halves (Fig. 2A, lane 8). In contrast, the point mutant c-myc–HSPC117 C122A was inactive as a tRNA ligase (Fig. 2A, lane 7) in immunoprecipitates containing equal amounts of wild-type and C122A mutant c-myc–HSPC117 (Fig. 2B and fig. S4).

Fig. 2

Affinity purification of c-myc–HSPC117 from stably transfected HeLa cell lines yields an RNA>p ligase. (A) Immunoprecipitates (IPs) of wild-type (WT) or C122A c-myc–HSPC117 were incubated with tRNA exon halves. An IP prepared from a non-expressing clone was used as negative control. Asterisks denote unrelated bands. (B) Detection of WT and mutant c-myc–HSPC117 in IPs by Western blot. (C) tRNA exon halves were incubated with recombinant CLP1 or T4 Pnkp in the presence or absence of ATP and used as a substrate for ligation with c-myc–HSPC117. (D) RNase T1 fragments derived from [α-32P]UTP-radiolabeled mature tRNA generated either by T4 Pnkp/Rnl1 or affinity-purified c-myc–HSPC117 were resolved by denaturing gel electrophoresis. RNA heptamers were isolated from the gel, digested with RNase T2, and analyzed by thin-layer chromatography (TLC). (E) Circular, [α-32P]ATP-radiolabeled intron generated either by T4 Pnkp/Rnl1 or affinity-purified c-myc–HSPC117 was isolated from gels, digested with nuclease P1, and analyzed by TLC.

RNA>p ligase requires a 5′-OH at the termini of its substrates. Accordingly, no ligase activity could be detected in c-myc–HSPC117 immunoprecipitates when tRNA exon halves were phosphorylated with the recombinant 5′-OH RNA kinase CLP1 (23) (Fig. 2C, compare lanes 2 and 3). Removing the 2′,3′-cyclic phosphate of the tRNA 5′ exon with bacteriophage T4 polynucleotide kinase (T4 Pnkp) abolished ligase activity of c-myc–HSPC117 immunoprecipitates (Fig. 2C, lanes 4 and 5). Thus, ligation by HSPC117 is dependent on 5′-OH (see also Fig. 1A) and 2′,3′-cyclic phosphate.

To test whether ligation by HSPC117 results in incorporation of the precursor-derived 2′,3′-cyclic phosphate into mature tRNA, we performed a nearest-neighbor analysis of the splice junction phosphate (12, 14) (fig. S1A, lower branch, and fig. S5). tRNA exon halves with a radiolabeled 2′,3′-cyclic phosphate at the terminus of the 5′-exon half were prepared from [α-32P]uridine triphosphate (UTP)–radiolabeled pre-tRNA (fig. S5A) and were ligated either with c-myc–HSPC117 immunoprecipitate or with a mixture of T4 Pnkp and T4 Rnl1 as a negative control. The precursor-derived 2′,3′-terminal phosphate, liberated as guanosine 3′-monophosphate (Gp) upon digestion of the splice junction of mature tRNA with ribonuclease (RNase) T2, was retained only upon ligation with c-myc–HSPC117 (Fig. 2D, lane 1) but not with T4 Pnkp/T4 Rnl1 (Fig. 2D, lane 2). A similar nearest-neighbor analysis was also carried out with [α-32P]adenosine triphosphate (ATP)–radiolabeled linear intron after its conversion to circularized intron by c-myc–HSPC117 immunoprecipitate (fig. S5B). Detection of radiolabeled uridine 5′-monophosphate (pU) in nuclease P1 digests of introns circularized by c-myc–HSPC117 but not by T4 Pnkp/Rnl1 supported retention of the splice junction phosphate (Fig. 2E, compare lanes 1 and 2). Therefore, HSPC117 joins tRNA exon halves by incorporating the precursor-derived splice junction phosphate into the mature tRNA as a canonical 3′,5′-phosphodiester, as previously shown (12, 13).

In an unrelated effort aiming to purify spliceosomal particles, we discovered that a complex containing HSPC117 was co-selected during immunoaffinity chromatography of human SF3b complexes (fig. S6A). SF3b is essential for pre-mRNA splicing and associates with the U2 small nuclear ribonucleoprotein (snRNP). SF3b was affinity-selected from HeLa nuclear extract depleted of spliceosomal snRNPs. During size exclusion chromatography of the affinity-selected proteins, in addition to the SF3b complex (fractions 12 to 18), a smaller complex peaking in fractions 23 to 26 was observed (Fig. 3A, left panel). This complex contained HSPC117 together with the DEAD-box helicase DDX1, CGI-99/C14ORF166, FAM98B, and two forms of ASW/C2ORF49 (Fig. 3A, right panel, and table S2). This set of proteins overlaps with the MS analysis results of immunoprecipitates of c-myc–HSPC117 from stable cell clones (table S3) and the proteins identified in the MonoQ RNA>p ligase fraction (table S1). Consistent with a role of an HSPC117 complex in RNA ligation, we were able to inhibit interstrand ligation and tRNA maturation reactions by addition of DNA duplexes reported to specifically bind to HSPC117, DDX1, and CGI-99 (24) (fig. S9). Fractions containing the HSPC117 complex, but not the SF3b complex, were sufficient to convert tRNA exon halves and linear intron into mature tRNA and circularized intron (Fig. 3B) and were able to rescue the tRNA maturation defect upon depletion of HSPC117 in cell extracts (fig. S6B). Ligation by the HSPC117 complex was strongly stimulated by the addition of ATP (25) (fig. S7). Because efficient depletion of the HSPC117-interacting proteins did not compromise interstrand ligation or tRNA maturation as severely as did silencing of HSPC117 itself (Fig. 3, C to F, and fig. S8), and because mutation of a single, highly conserved cysteine residue of HSPC117 abolishes its RNA ligase activity (Fig. 2A and fig. S4), we conclude that HSPC117 is the only essential subunit of the ligase complex. It is noteworthy that the Pyrobaculum aerophilum RtcB protein was recently identified as a monomeric archaeal RNA>p ligase (26).

Fig. 3

An active RNA>p ligase complex is co-selected with SF3b monoclonal antibodies. (A) Size exclusion chromatography of SF3b and HSPC117 complexes after affinity co-selection. Fractions were analyzed by SDS–polyacrylamide gel electrophoresis. (B) tRNA exon half ligation assay of eluted fractions. Asterisks denote unrelated bands. (C and D) Extracts prepared from cells depleted of HSPC117, DDX1, CGI-99, FAM98B, ASW, and EGFP as a control were assayed for interstrand ligation (C) and tRNA maturation (D). (E) Confirmation of the depletion of HSPC117, DDX1, CGI-99, FAM98B, and ASW mRNAs by quantitative polymerase chain reaction (PCR). Results are depicted as means ± SD of triplicate PCR reactions. (F) Western blot for HSPC117, DDX1, FAM98B, and β-actin as a loading control.

In living cells, interstrand ligation was dependent on HSPC117 (Fig. 4A, compare lanes 4 and 7 with lanes 3 and 6, respectively) and RNA 3′-P terminal cyclase (RTCD1) (Fig. 4A, compare lanes 5 and 8 with lanes 3 and 6, respectively). To test the effect of silencing of HSPC117 on processing of de novo synthesized pre-tRNA, we followed the fate of inducible pre-tRNA reporter transcripts in living cells. Induced transcription of intron-containing reporter pre-tRNAs distinguishable from endogenous pre-tRNAs (fig. S10A) revealed a delay in the formation of mature tRNA in cultured cells depleted of HSPC117 (Fig. 4B, compare lanes 1 to 3 with lanes 4 to 6 and lanes 7 to 9 with lanes 10 to 12, and Fig. 4C). These data establish a role for HSPC117 as an RNA ligase with broad substrate specificity and with a function in tRNA processing in living cells.

Fig. 4

Silencing of HSPC117 abolishes interstrand ligation and partially impairs tRNA processing in living cells. (A) Ligation of radiolabeled 3′-P, 5′-OH dsRNAs upon transfection into HeLa cells pretransfected with siRNAs targeting HSPC117, RTCD1, or EGFP as a control gene was monitored by denaturing gel electrophoresis. (B) Tet repressor–expressing HeLa cells were cotransfected with siRNAs targeting HSPC117 or EGFP and reporter constructs encoding Tet-inducible pre-tRNA IleUAU (lanes 1 to 6) or pre-tRNA ScPhe (lanes 7 to 12). After induction, RNA was isolated at indicated time points and analyzed by Northern blot. (C) The signals corresponding to mature tRNA were quantified by phosphorimaging. Values were normalized to U6 hybridization signals; the signals obtained in EGFP siRNA treated cells at 48 hours were arbitrarily set to 100%. Values are depicted as means ± SD; P values were obtained by unpaired t test (N = 3).

Both RNA>p ligase and T4 Rnl1–like ligation mechanisms have been detected in human cells (12, 27) but RNA>p ligase seems to play a dominant role in human tRNA splicing (12, 28). We have identified HSPC117 as an essential component of the prevalent human tRNA splicing pathway. Recently, HSPC117 or the RNA>p ligase pathway have also been implicated in RNA processing during viral replication (29, 30). The high degree of conservation of HSPC117/RtcB proteins is suggestive of shared roles for this protein family in organisms as distantly related as humans and E. coli.

Supporting Online Material

www.sciencemag.org/cgi/content/full/331/6018/760/DC1

Materials and Methods

Figs. S1 to S10

Tables S1 to S3

References

References and Notes

  1. See supporting material on Science Online.
  2. EBI InterPro database entry www.ebi.ac.uk/interpro/IEntry?ac=IPR001233.
  3. We thank H. Beier, T. Biederer, T. Clausen, A. Meinhart, A. F. Nielsen, R. Schroeder, K. Sheppard, T. Tuschl, and S. Westermann for reagents and helpful discussions; and G. Heyne, O. Kuzyk, G. Schmauss, and G. Stengl for technical assistance; and H. Urlaub for MS analysis of HSPC117 complexes. M.E. was a Feodor-Lynen Postdoctoral Fellow of the Alexander von Humboldt Stiftung (Bonn, Germany). Supported by grants from the European Commission (EURASNET-518238) (R.L.), the Fonds zur Förderung der wissenschaftlichen Forschung (W1207 RNA Biologie) (J.P.), and the National Institute of General Medical Sciences (D.S.). J.M. would like to especially acknowledge the generous financial contribution and support by S.D. Prinz Max von und zu Liechtenstein.
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