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RNA. Jun 2008; 14(6): 1214–1227.
PMCID: PMC2390804

Competition between the Rex1 exonuclease and the La protein affects both Trf4p-mediated RNA quality control and pre-tRNA maturation

Abstract

Although nascent noncoding RNAs can undergo maturation to functional RNAs or degradation by quality control pathways, the events that influence the choice of pathway are not understood. We report that the targeting of pre-tRNAs and certain other noncoding RNAs for decay by the TRAMP pathway is strongly influenced by competition between the La protein and the Rex1 exonuclease for access to their 3′ ends. The La protein binds the 3′ ends of many nascent noncoding RNAs, protecting them from exonucleases. We demonstrate that unspliced, end-matured, partially aminoacylated pre-tRNAs accumulate in yeast lacking the TRAMP subunit Trf4p, indicating that these pre-tRNAs normally undergo decay. By comparing RNA extracted from wild-type and mutant yeast strains, we show that Rex1p is the major exonuclease involved in pre-tRNA trailer trimming and may also function in nuclear CCA turnover. As the accumulation of end-matured pre-tRNAs in trf4Δ cells requires Rex1p, these pre-tRNAs are formed by exonucleolytic trimming. Accumulation of truncated forms of 5S rRNA and SRP RNA in trf4Δ cells also requires Rex1p. Overexpression of the La protein Lhp1p reduces both exonucleolytic pre-tRNA trimming in wild-type cells and the accumulation of defective RNAs in trf4Δ cells. Our experiments reveal that one consequence of Rex1p-dependent 3′ trimming is the generation of aberrant RNAs that are targeted for decay by TRAMP.

Keywords: RNA quality control, tRNA maturation, exonucleases, La protein, TRAMP, Rex1p

INTRODUCTION

The majority of noncoding RNAs in eukaryotic cells are synthesized as precursors that must be processed to mature RNAs or degraded by quality control pathways. However, little is known of the mechanisms that influence whether newly synthesized noncoding RNAs are matured to produce functional RNAs or distinguished as aberrant. In the yeast Saccharomyces cerevisiae, a nuclear complex called TRAMP polyadenylates the 3′ ends of certain defective noncoding RNAs and assists their decay by a 3′ to 5′ exoribonuclease, the nuclear exosome (Kadaba et al. 2004; LaCava et al. 2005; Vanacova et al. 2005; Wyers et al. 2005; Davis and Ares 2006). Although purified TRAMP preferentially polyadenylates the misfolded forms of two mature tRNAs over the correctly folded RNAs (Vanacova et al. 2005; Schneider et al. 2007), the structural features that confer TRAMP recognition have not been elucidated. In vivo, the only known tRNA substrate of TRAMP, hypomethylated tRNAi Met, is recognized as a 3′-trailer-containing pre-tRNA (Kadaba et al. 2006). However, the 3′ ends of pre-tRNAs, pre-5S rRNAs, the SRP RNA, and many other nascent noncoding RNAs are normally bound by the La protein (Wolin and Cedervall 2002), which could block polyadenylation by TRAMP. La protects the 3′ ends of nascent RNAs from exonucleases and also assists folding of certain pre-tRNAs (Chakshusmathi et al. 2003).

The 3′-end maturation of pre-tRNAs is particularly complex, as maturation involves multiple steps and can take place by more than one pathway. Studies of tRNA maturation in budding and fission yeasts have resulted in a model in which La sequesters pre-tRNA 3′ ends from exonucleases, favoring trailer removal by an endonuclease (Van Horn et al. 1997; Yoo and Wolin 1997; Huang et al. 2006). Although the identity of the endonuclease that matures the trailer in yeast has not been reported, work in other organisms has revealed that pre-tRNA trailers are removed by the endonuclease RNase Z (Redko et al. 2007; Spath et al. 2007). In cells lacking La, unidentified exonucleases contribute to 3′ maturation (Van Horn et al. 1997; Yoo and Wolin 1997). After end maturation and CCA addition, aminoacylation occurs within nuclei (Lund and Dahlberg 1998; Sarkar et al. 1999; Grosshans et al. 2000). How quality control pathways intersect with these maturation pathways is unknown. Following end maturation and nuclear export, splicing of intron-containing pre-tRNAs occurs in the cytoplasm (for review, see Hopper 2006).

Here we investigate how the La protein interfaces with 3′ exonucleases and TRAMP to influence RNA fate in Saccharomyces cerevisiae. We report that the targeting of certain pre-tRNAs and other noncoding RNAs for degradation by TRAMP is influenced both by La protein binding and by 3′ trimming by the nonexosome exonuclease Rex1p. We show that unspliced, end-matured, partially aminoacylated pre-tRNAs accumulate in S. cerevisiae cells lacking Trf4p, the catalytic subunit of TRAMP. We provide evidence that Rex1p is the major exonuclease involved in pre-tRNA maturation and nuclear CCA turnover. The accumulation of unspliced pre-tRNAs in trf4Δ cells requires Rex1p, indicating that the defective pre-tRNAs arise as a consequence of Rex1p trimming. As overexpression of the La protein Lhp1p reduces both exonucleolytic pre-tRNA maturation in wild-type cells and the accumulation of unspliced pre-tRNAs in cells lacking Trf4p, Lhp1p likely competes with Rex1p for access to pre-tRNA 3′ ends. The accumulation of truncated forms of 5S rRNA and SRP RNA in trf4Δ cells also depends on Rex1p. Thus, one consequence of 3′-end trimming by Rex1p is the generation of defective RNAs that are TRAMP substrates.

RESULTS

A pre-tRNASer CGA maturation intermediate accumulates in trf4Δ cells but is less abundant in cells that also lack Lhp1p

To determine if the binding of Lhp1p to nascent RNAs affects the spectrum of RNAs targeted for decay by TRAMP, we compared RNA from strains lacking LHP1, TRF4, and also RRP6, which encodes an exoribonuclease that is part of the nuclear exosome. Northern analyses, followed by quantitation, revealed that an intron-containing form of pre-tRNASer CGA increased severalfold in trf4Δ cells (Fig. 1A, top panel, lane 2; also Fig. 1B) but was present in lower amounts in trf4Δ lhp1Δ strains (Fig. 1A, lane 4). In trf4Δ rrp6Δ strains, this pre-tRNA species increased more significantly (Fig. 1A, top panel, lane 6). Probing to detect the mature tRNA revealed that tRNASer CGA increased approximately 1.5-fold in trf4Δ cells and by approximately threefold in trf4Δ rrp6Δ cells (Fig. 1A, second panel, lanes 2,6; also Fig. 1B). The finding that both the precursor and mature tRNASer CGA increase in trf4Δ and trf4Δ rrp6Δ strains indicates that a fraction of the precursors are normally degraded through the quality control pathway involving TRAMP and the nuclear exosome.

FIGURE 1.
Several noncoding RNAs accumulate in trf4Δ cells. (A) RNA extracted from the indicated strains was subjected to Northern blotting to detect precursor and mature forms of tRNASer CGA, tRNAArg CCG, U4 and U6 snRNAs, SRP RNA, U3 snoRNA, and 5S rRNA. ...

The pre-tRNASer CGA that accumulates in trf4Δ cells was similar in size to the end-matured, intron-containing pre-tRNA in wild-type and lhp1Δ cells. As described (Yoo and Wolin 1997), three intron-containing forms of pre-tRNASer CGA are detected in wild-type cells: a primary transcript with 5′ and 3′ extensions, an intermediate with a mature 5′ end and a 3′ extension, and a pre-tRNA with mature 5′ and 3′ ends (Fig. 1A, lane 1). In lhp1Δ cells, the 3′ end is initially nibbled by exonucleases, with digestion stopping at a stem formed by base-pairing of the leader and trailer. Thus, the leader- and trailer-containing RNA is shorter and more heterogeneous than in wild-type cells (Fig. 1A, lanes 3,4,7,8). Following cleavage of the leader by RNase P, exonucleases generate the mature 3′ end. As a result, a discrete processing intermediate containing a mature 5′ end and a 3′ trailer is not detected in lhp1Δ cells (Yoo and Wolin 1997). We confirmed the identity of the pre-tRNA that accumulates in trf4Δ cells by probing blots with oligonucleotides specific for the leader and trailer (data not shown).

We also examined the levels of several other noncoding RNAs, including tRNAArg CCG (which is not made as an intron-containing precursor), the spliceosomal U4 and U6 snRNAs, SRP RNA, and the U3 snoRNA. PhosphorImager quantitation revealed that the levels of tRNAArg CCG and U6 RNA increased very slightly in trf4Δ cells and by approximately twofold in trf4Δ rrp6Δ cells (Fig. 1A; data not shown). Thus, as previously reported for U6 RNA (Wyers et al. 2005), a small fraction of these RNAs or their precursors may be polyadenylated by TRAMP and degraded by the exosome. In addition, a degradation fragment of the SRP RNA was detectable in trf4Δ cells and was more apparent in trf4Δ rrp6Δ cells (Fig. 1A, lanes 2,6,8). Finally, the intron-containing pre-U3 snoRNA, which is undetectable in wild-type cells, was easily detected in trf4Δ strains (Fig. 1A, lanes 2,4,6,8). Whether the accumulation of the pre-U3 snoRNA in trf4Δ cells represents a defect in splicing or a failure to degrade unspliced RNAs is unknown.

We determined whether the presence of Lhp1p impacts the growth of trf4Δ and rrp6Δ cells. Cells lacking TRF4 grow poorly at all temperatures (Wyers et al. 2005), while rrp6Δ cells grow poorly at 37°C (Briggs et al. 1998). Interestingly, we found that trf4Δ lhp1Δ cells grew significantly better than trf4Δ cells (Fig. 1E). Also, while the growth of rrp6Δ lhp1Δ cells was similar to that of rrp6Δ cells, longer incubations of the plates revealed that rrp6Δ trf4Δ lhp1Δ cells grew slightly better at 37°C than rrp6Δ trf4Δ cells (Fig. 1C). Because there are numerous changes in RNA metabolism in trf4Δ cells (LaCava et al. 2005; Wyers et al. 2005; Egecioglu et al. 2006; Kadaba et al. 2006), it is unclear which alterations contribute to the growth defects. Nonetheless, the finding that less unspliced pre-tRNASer CGA is detected in trf4Δ strains when LHP1 is deleted (Fig. 1A, cf. lanes 2 and 4), coupled with the finding that deletion of LHP1 partly alleviates the growth defect of trf4Δ cells, indicates that binding by Lhp1p to some nascent noncoding RNAs influences targeting by TRAMP.

Many intron-containing, end-matured pre-tRNAs accumulate in cells lacking Trf4p

Since the most striking alteration we detected in trf4Δ cells was the accumulation of the intron-containing pre-tRNASer CGA we determined if this was a general occurrence by examining other pre-tRNAs. Probing for seven other intron-containing pre-tRNAs (tRNASer GCU, tRNATyr GUA, tRNALys UUU, tRNAPhe GAA, tRNALeu UAG, tRNALeu CAA, and tRNAPro UGG) revealed that in all cases similar processing intermediates accumulated in trf4Δ stains, but were less abundant in trf4Δ lhp1Δ strains (Fig. 2A; data not shown). Fractionation in high-resolution sequencing gels revealed that the largest end-matured unspliced pre-tRNAs that accumulate in trf4Δ cells migrated slightly slower than their counterparts in wild-type cells (Fig. 2B, tRNALys UUU).

FIGURE 2.
Many end-matured unspliced pre-tRNAs accumulate in trf4Δ cells. (A) RNA extracted from wild-type, trf4Δ, lhp1Δ, and trf4Δ lhp1Δ cells were subjected to Northern blotting to detect the indicated intron-containing ...

Since yeast contain a second nuclear poly(A) polymerase, Trf5p, that is related in sequence and function to Trf4p (Egecioglu et al. 2006; Houseley and Tollervey 2006), we examined the levels of unspliced pre-tRNAs in trf5Δ cells. Pre-tRNA levels in trf5Δ and rrp6Δ trf5Δ cells were similar to wild-type cells (Fig. 2C, lanes 1,2,6). Thus, Trf5p does not play a major role in the decay of intron-containing pre-tRNAs.

One possible explanation for the finding that end-matured unspliced pre-tRNAs accumulate in trf4Δ cells but decrease in trf4Δ lhp1Δ cells is that Lhp1p stabilizes these pre-tRNAs against exonucleases. Immunoprecipitation with anti-Lhp1p antibodies revealed that, as described (Yoo and Wolin 1997), Lhp1p binds pre-tRNAs containing the trailer but does not bind the end-matured pre-tRNAs (Fig. 2D, lanes 5,7,8). Thus, the increased levels of these RNAs in trf4Δ cells compared to trf4Δ lhp1Δ cells are not due to protection of the end-matured pre-tRNAs by bound Lhp1p.

The nonexosome exonuclease Rex1p functions in tRNA 3′-end maturation

The finding that the end-matured unspliced pre-tRNAs are not bound by Lhp1p suggested that the presence of Lhp1p affects their fate at a step prior to 3′-end maturation. Since bound Lhp1p impedes access of 3′ to 5′ exonucleases (Yoo and Wolin 1997), one possibility was that binding of Lhp1p to the nascent pre-tRNAs influences the mechanism of 3′-trailer removal. For example, the presence of Lhp1p could influence the choice of maturation pathway (i.e., endonucleolytic versus exonucleolytic maturation) or could affect which exonucleases participate in maturation.

Compared to prokaryotes, where both the endonuclease RNase Z and multiple exonucleases contribute to pre-tRNA maturation (Redko et al. 2007), the pathway of pre-tRNA 3′-end maturation is poorly understood in eukaryotes. Although RNase Z was shown to contribute to pre-tRNA maturation in Drosophila melanogaster (Dubrovsky et al. 2004), the exonucleases that mature eukaryotic pre-tRNAs have not been identified. Comparison of the tRNASer CGA processing intermediates in lhp1Δ and rrp6Δ lhp1Δ cells revealed that while their levels increased slightly in rrp6Δ lhp1Δ cells, the pattern of intermediates was unchanged (Fig. 1A, lanes 3,7; Fig. 2C, lanes 3,7). Thus, while Rrp6p may contribute to 3′-end maturation in lhp1Δ cells, other exonucleases must be involved.

We examined whether nonexosome exonucleases contribute to pre-tRNA maturation. Three of these nucleases, Rex1p, Rex2p, and Rex3p, function in noncoding RNA processing (van Hoof et al. 2000). Rex1p is required for the 3′ maturation of a tRNAArg UCU that is the only yeast tRNA transcribed as the 5′ cistron in a dicistronic tRNA (van Hoof et al. 2000). (After separation of the downstream pre-tRNA by RNase P cleavage, pre-tRNAArg UCU does not contain the 3′ uridylates required for Lhp1p binding.) Rex1p also functions in 5S rRNA maturation, and Rex2p is required for U4 3′ trimming, while Rex3p functions in maturation of MRP RNA. Moreover, Rex1p and Rex2p function redundantly in 5.8S rRNA maturation, while Rex1p, Rex2p, and Rex3p function redundantly in maturing the spliceosomal U5 snRNA (van Hoof et al. 2000). A fourth nuclease, Rex4p, has been implicated in rRNA maturation (Eppens et al. 2002).

To examine the role of these nucleases, RNA was extracted from rex1Δ, rex2Δ, and rex3Δ strains and subjected to Northern analyses. Probing to detect pre-tRNASer CGA revealed that the overall pattern of pre-tRNAs in these strains was similar to the wild-type strain (Fig. 3A, lanes 1,3,5,7). However, in rex1Δ lhp1Δ cells, the pattern of tRNASer CGA precursors differed from that of lhp1Δ cells, in that the two 3′-trailer-containing pre-tRNAs that are characteristic of wild-type cells became detectable (Fig. 3A, lane 4). Thus, Rex1p participates in pre-tRNASer CGA 3′-end trimming in lhp1Δ cells.

FIGURE 3.
Rex1p functions in pre-tRNA maturation. (A,B) RNAs from the indicated strains were subjected to Northern blotting to detect precursor and mature forms of (top) tRNASer CGA, (middle) tRNATyr GUA, and (bottom) tRNALys UUU. As a loading control, blots were ...

Interestingly, probing for pre-tRNATyr GUA and pre-tRNALys UUU revealed that Rex1p also functions in 3′-end trimming in wild-type cells. In rex1Δ cells, the pre-tRNATyr GUA containing a mature 5′ end and a 3′ trailer became more prominent than in wild-type cells (Fig. 3A, lane 3, indicated by a dot). For pre-tRNALys UUU, the precursor containing the leader and trailer is identical in length between wild-type and lhp1Δ cells, and the intermediate containing a mature 5′ end and the trailer is undetectable (Fig. 3A, lanes 1,2). Strikingly, in rex1Δ cells, the form containing the leader and the trailer is longer, and a pre-tRNA containing a mature 5′ end and the trailer becomes apparent (Fig. 3A, lane 3, dot). Both these pre-tRNAs are slightly shorter in rex1Δ lhp1Δ cells (Fig. 3A, lane 4). One explanation is that in wild-type cells, a fraction of both pre-tRNATyr GUA and pre-tRNALys UUU undergoes 3′-end trimming by Rex1p. In rex1Δ cells, these pre-tRNAs are stabilized by Lhp1p binding, while in rex1Δ lhp1Δ cells, other nucleases access the 3′ ends.

Rrp6p contributes to pre-tRNA maturation in lhp1Δ cells

Although our experiments revealed that Rex1p participates in tRNA maturation in lhp1Δ cells, the pre-tRNAs in rex1Δ lhp1Δ cells were more heterogeneous and were present at lower levels than in wild-type cells, suggesting that additional nucleases contribute. Since Rex1p, Rex2p, and Rex3p have overlapping functions in RNA processing (van Hoof et al. 2000), we analyzed RNA from cells lacking combinations of the nucleases. In all cells lacking both Rex1p and Lhp1p, the primary transcript and the two processing intermediates characteristic of wild-type cells were detected (Fig. 3B, lanes 4,6,10). However, the levels of these precursors did not increase when Rex2p, Rex3p, and Rex4p were also deleted (Fig. 3B; data not shown), suggesting that these nucleases do not play a major role in pre-tRNA maturation in lhp1Δ cells.

We examined whether Rrp6p functions with Rex1p in pre-tRNA maturation. Although rrp6Δ rex1Δ cells were reported to be inviable (van Hoof et al. 2000), in our strain background this combination was viable but extremely slow growing (Fig. 4A). Thus, as previously noted (van Hoof et al. 2000), Rex1p and Rrp6p may function redundantly in the processing of at least one RNA. However, comparison of RNA from wild-type and rex1Δ rrp6Δ cells did not reveal differences in pre-tRNA maturation intermediates or mature tRNAs (Fig. 4B, lanes 1,5), indicating that a failure to mature tRNAs does not account for the synthetic growth defect.

FIGURE 4.
Rrp6 and Rex1p participate in pre-tRNA maturation in lhp1Δ cells. (A) Fivefold serial dilutions of the indicated strains were spotted on YPD agar and grown for 3 d at 25°C. (B) RNA from the indicated strains were subjected to Northern ...

We examined whether Rrp6p and Rex1p function redundantly to mature tRNAs in lhp1Δ cells. In rrp6Δ rex1Δ lhp1Δ cells, the levels of pre-tRNASer CGA and pre-tRNATyr GUA primary transcripts and maturation intermediates were similar to wild-type cells (Fig. 4B, lane 6). As deleting both Rex1p and Rrp6p restores the pattern of pre-tRNA maturation intermediates in lhp1Δ cells to that of wild-type cells (Fig. 4B, cf. lanes 1,2,6), we conclude that both Rex1p and Rrp6p contribute to pre-tRNA 3′ maturation in lhp1Δ cells. However, as the levels of mature tRNA are unchanged in rrp6Δ rex1Δ lhp1Δ cells, other nucleases, such as the endonuclease RNase Z, may carry out tRNA maturation in the absence of these exonucleases.

Accumulation of intron-containing pre-tRNAs in trf4Δ cells requires Rex1p

To determine if the accumulation of end-matured unspliced pre-tRNAs in trf4Δ cells was a consequence of exonucleolytic 3′-end trimming, we examined trf4Δ rex1Δ strains. As previously noted for rex1Δ cells, the 3′-trailer-containing forms of pre-tRNATyr GUA and pre-tRNALys UUU were increased in abundance in trf4Δ rex1Δ cells compared to wild-type cells (Fig. 5A, lane 5, dots). However, the levels of the end-matured pre-tRNAs in these cells were similar to wild-type cells (Fig. 5A, cf. lanes 1 and 5). Thus, Rex1p is required for the accumulation of end-matured unspliced pre-tRNAs in trf4Δ cells. However, in trf4Δ rex1Δ lhp1Δ strains, the levels of the end-matured unspliced pre-tRNAs increased slightly compared to trf4Δ rex1Δ cells (Fig. 5A, cf. lanes 5 and 7). One explanation for this result is that in the absence of both Lhp1p and Rex1p, 3′-maturation by other exonucleases (such as Rrp6p) contributes to the accumulation of end-matured unspliced pre-tRNAs in trf4Δ cells. Multiple attempts to isolate trf4Δ rex1Δ rrp6Δ strains were unsuccessful, suggesting that this combination is inviable.

FIGURE 5.
Rex1p is required for the accumulation of aberrant RNAs in trf4Δ cells. (A) RNA from the indicated strains was fractionated in 8% polyacrylamide, 8.3 M urea gels and subjected to Northern blotting to detect precursor and mature forms of (top) ...

Our finding that the accumulation of end-matured pre-tRNAs in trf4Δ cells requires Rex1p was consistent with a model in which Rex1p and Lhp1p compete for access to pre-tRNA 3′ ends. In this scenario, those pre-tRNAs that fail to bind Lhp1p would undergo trimming by Rex1p. To test this hypothesis, we examined whether overexpression of LHP1 would decrease the fraction of end-matured unspliced pre-tRNAs that accumulate in trf4Δ cells. Wild-type and trf4Δ cells were transformed with a low copy plasmid containing LHP1. Although the levels of pre-tRNASer CGA and pre-tRNALys UUU were unchanged, the pre-tRNATyr GUA containing a mature 5′ end and the trailer became more prominent when Lhp1p was overexpressed in wild-type cells (Fig. 5B, lane 2, indicated by the dot). Consistent with the idea that Lhp1p and Rex1p compete for pre-tRNA ends, this change in the pattern of pre-tRNATyr GUA intermediates was similar to that detected in rex1Δ cells (Fig. 4A, lanes 1,3, dots; 5A, lanes 1,3, dots). Most strikingly, when LHP1 was overexpressed in trf4Δ strains, the end-matured forms of all three pre-tRNAs decreased to wild-type levels (Fig. 5B, lane 4). Since Rex1p is required for the accumulation of these RNAs (Fig. 5A), our results suggest that Lhp1p competes with Rex1p for access to pre-tRNA 3′ ends.

We note that these results do not explain the finding that less end-matured pre-tRNAs accumulate in trf4Δ lhp1Δ cells (Figs. 1A, ,2A).2A). Although Rex1p is the major exonuclease that trims pre-tRNA 3′ ends in cells containing Lhp1p, additional exonucleases, such as Rrp6p, access nascent pre-tRNAs in lhp1Δ cells (Fig. 4B). One possibility is that 3′ trimming by these other exonucleases results in fewer RNAs that are TRAMP targets (see Discussion).

Rex1p is required for the accumulation of truncated forms of SRP RNA and 5S rRNA in trf4Δ cells

The finding that the end-matured pre-tRNAs that accumulate in trf4Δ cells result from Rex1p-dependent trimming prompted us to ask if other aberrant RNAs that are TRAMP substrates are generated by Rex1p. Since a truncated form of the SRP RNA accumulates in trf4Δ cells (Fig. 1A, lane 2), we determined if accumulation of this RNA requires Rex1p. The truncated RNA was undetectable in trf4Δ rex1Δ cells (Fig. 5C, lane 5), indicating that Rex1p is required for its formation. This was surprising because although nascent SRP RNA is bound by Lhp1p (Yoo and Wolin 1994), this RNA is not made as a 3′ extended precursor and has not been described to undergo 3′ trimming (Felici et al. 1989). Consistent with competition between Lhp1p and Rex1p, the truncated RNA was not detected when LHP1 was overexpressed in trf4Δ cells (Fig. 5C, lane 11).

Another aberrant RNA that accumulates in trf4Δ cells is a 3′ truncated form of 5S rRNA that is detected on long exposures of Northern blots (Kadaba et al. 2006). Similar to the SRP RNA, the truncated RNA was detected in trf4Δ cells (Fig. 5C, lane 2), but not in trf4Δ rex1Δ cells (Fig. 5C, lane 5). However, the truncated RNA remained detectable in trf4Δ cells when LHP1 was present on the low copy plasmid (Fig. 5C, lanes 10,11), indicating that the resulting two- to threefold increase in Lhp1p was insufficient to block access of Rex1p to the RNA. Since 5S rRNA is normally made as a 3′-extended precursor that undergoes Rex1p trimming (van Hoof et al. 2000), the truncated 5S RNA that accumulates in trf4Δ cells may be formed by Rex1p that fails to disengage at the mature 3′ end. Taken together with our finding that accumulation of the truncated SRP RNA requires Rex1p, we conclude that one consequence of Rex1p-dependent trimming is the generation of aberrant RNAs that are TRAMP targets.

Rex1p may also function in nuclear CCA turnover

Interestingly, fractionation of RNA from rex1Δ cells in high-resolution gels revealed that for pre-tRNASer CGA and pre-tRNATyr GUA, but not pre-tRNALys UUU, the longest end-matured pre-tRNAs migrated more slowly than the wild-type RNAs (Fig. 6A). This was most obvious for pre-tRNATyr GUA, where four bands corresponding to end-matured pre-tRNAs were detected in rex1Δ cells, compared to three in wild-type cells (Fig. 6A, lanes 3,4, dots). Since longer end-matured pre-tRNAs also accumulate in trf4Δ cells (Fig. 2B), we determined their sizes by comparing their migration in denaturing gels with pre-tRNAs from cells carrying a temperature-sensitive mutation in RNA1, which encodes the Ran GTPase-activating protein that is required for nuclear export (Corbett et al. 1995). In rna1-1 cells at 37°C, intron-containing pre-tRNAs accumulate that contain CCA (Hopper et al. 1978; Knapp et al. 1978). The longest pre-tRNAs in rex1Δ and trf4Δ cells were identical in size to the longest pre-tRNAs in rna1-1 cells at 37°C (Fig. 6B). These pre-tRNAs were also ~3 nt longer than the shortest pre-tRNAs in cells containing a temperature-sensitive mutation in the CCA-adding enzyme (cca1-1) (Aebi et al. 1990).

FIGURE 6.
Pre-tRNAs that accumulate in trf4Δ and rex1Δ cells contain CCA. (A) RNA from wild-type, lhp1Δ, rex1Δ, and rex1Δ lhp1Δ cells was fractionated in a high-resolution gel and subjected to Northern blotting to ...

To determine if the longer forms of pre-tRNATyr GUA and pre-tRNASer CGA that accumulate in rex1Δ and trf4Δ cells contained trailer sequences or CCA, we examined whether they could be extended with T7 DNA polymerase and oligonucleotide splints complementary to either the CCA-containing pre-tRNAs or the trailer-containing RNAs. In this technique (Hausner et al. 1990), the pre-tRNA will only be extended if the oligonucleotide is exactly complementary to the 3′ end. To assay CCA addition, oligonucleotides were designed that would extend both CC- and CCA-containing pre-tRNAs. A second set of oligonucleotides was designed to extend pre-tRNAs containing either a partly or fully cleaved trailer.

Total RNA from wild-type, rex1Δ, trf4Δ, and rna1-1 cells was incubated with the oligonucleotides and extended with T7 DNA polymerase. In the presence of oligonucleotides that extend pre-tRNAs containing trailer sequences, the two shortest end-matured forms of pre-tRNATyr GUA in all strains were replaced by extended species (Fig. 6C, lanes 2,5,8,11, open circles). In the presence of oligonucleotides that extend pre-tRNAs containing CC- or CCA, the two largest forms of the end-matured pre-tRNAs were replaced by extended RNAs (Fig. 6C, lanes 3,6,9,12, diamonds). Based on migration in denaturing gels and the ability of these pre-tRNAs to be extended with oligonucleotides, we conclude that the largest end-matured unspliced pre-tRNATyr GUA species in wild-type cells contain -CC at their 3′ ends (Fig. 6C, lane 3), while the slightly longer pre-tRNAs that accumulate in rex1Δ, trf4Δ, and rna1-1 cells contain CCA (Fig. 6C, lanes 6,9,12). Similar results were obtained using oligonucleotides specific for pre-tRNASer CGA (data not shown).

The finding that CC-containing forms of pre-tRNATyr GUA and pre-tRNASer CGA predominate in wild-type cells, while CCA-containing forms of these pre-tRNAs accumulate in rex1Δ cells, suggests that Rex1p may contribute to the turnover of CCA. In Escherichia coli, where CCA turnover has been best characterized, a subset of tRNAs cycle between an aminoacylated form that is resistant to exonucleases and a deacylated form in which the CCA is converted to CC- by RNase T and then repaired by the CCA-adding enzyme (Deutscher 2003). Since Rex1p is a nuclear protein (Frank et al. 1999) and we have not detected differences in the CCA ends of mature tRNAs (data not shown), this role of Rex1p may be confined to nuclei. Consistent with a role that is not dependent on the choice of end maturation pathway, the CCA-containing pre-tRNAs that accumulate in rex1Δ cells are present at similar levels in rex1Δ lhp1Δ cells (Fig. 6A) and are unchanged on Lhp1p overexpression (data not shown). A model for the role of Rex1p in tRNA maturation and CCA turnover is shown in Figure 7.

FIGURE 7.
Model for pre-tRNA 3′-end maturation. (A) In wild-type cells, Lhp1p protects the 3′ ends of many nascent pre-tRNAs from exonucleases. Thus, the first cleavage is by RNase P, resulting in an Lhp1p-bound pre-tRNA with a mature 5′ ...

Some unspliced pre-tRNAs in trf4Δ cells undergo aminoacylation

The finding that the longest end-matured pre-tRNAs in trf4Δ cells contain CCA, while the longest pre-tRNAs detected in wild-type cells contain -CC, suggested that some pre-tRNAs in trf4Δ cells might be aminoacylated, since the presence of the amino acid should prevent end turnover. To examine charging, RNA from wild-type and trf4Δ strains was extracted under acidic conditions to stabilize aminoacyl-tRNA linkages (Varshney et al. 1991). Half of each sample was treated with base to remove the amino acid prior to fractionation in acidic acrylamide gels. For most intron-containing pre-tRNAs, only one form of the end-matured precursor was detected and its migration was unaffected by base treatment (data not shown). Thus, it was unclear if the pre-tRNAs were uncharged or if their large size precluded separation of the aminoacylated and deacylated forms. However, the shortest end-matured intron-containing pre-tRNA, pre-tRNATyr GUA, migrated as a doublet in trf4Δ cells (Fig. 6D, lane 3). Consistent with aminoacylation, incubation with base largely eliminated the slower-migrating species (Fig. 6D, lane 4). PhosphorImager quantitation revealed that ~23% of the end-matured pre-tRNA in trf4Δ cells was aminoacylated. In contrast, aminoacylated intron-containing pre-tRNAs were not detected in wild-type cells (Fig. 6D, lanes 1,2).

DISCUSSION

Although it is now accepted that cells possess quality control mechanisms to degrade defective noncoding RNAs, the factors that influence whether newly synthesized RNAs will undergo maturation and assembly with proteins or will be targeted for decay remain poorly understood. We found that end-matured, intron-containing pre-tRNAs and a truncated SRP RNA accumulate in trf4Δ cells, indicating these RNAs are normally targeted for degradation. We demonstrated that Rex1p is the major exonuclease involved in trimming pre-tRNA trailers and may also function in nuclear turnover of CCA. As the accumulation of both the unspliced pre-tRNAs and two truncated RNAs in trf4Δ cells requires Rex1p, these defective RNAs likely form as a result of exonucleolytic trimming. Since most of these RNAs do not accumulate when Lhp1p is overexpressed in trf4Δ cells, Lhp1p likely competes with Rex1p for access to the 3′ ends of nascent RNAs.

Pre-tRNA maturation

Our findings, together with previous studies (Evans and Engelke 1990; Furter et al. 1992; Yoo and Wolin 1997; van Hoof et al. 2000), indicate that exonucleases and at least one endonuclease contribute to pre-tRNA maturation. In prokaryotes, exonucleolytic maturation is largely confined to tRNAs in which the CCA is encoded, while RNase Z carries out maturation of most pre-tRNAs that lack CCA. However, the preference for endonucleolytic maturation of CCA-less pre-tRNAs may be largely due to the presence of downstream terminator structures that constitute a barrier for exonucleases (Redko et al. 2007). Consistent with this hypothesis, several Bacillus subtilis CCA-less pre-tRNAs with short trailers are processed correctly when RNase Z is depleted (Pellegrini et al. 2003). In eukaryotes, trailers are largely short and unstructured, with La acting as an exonuclease barrier. Our result that end-matured pre-tRNAs accumulate in trf4Δ cells, coupled with the finding that Rex1p is required for their accumulation, indicates that many pre-tRNAs undergo at least some trimming by Rex1p. However, as the levels of mature tRNAs are unaffected in rex1Δ cells, we do not know what fraction of the pre-tRNAs trimmed by Rex1p become functional mature tRNAs.

Our results, together with those of van Hoof et al. (2000), suggests that Rex1p may be functionally equivalent to RNase T, the only E. coli exoribonuclease that efficiently removes nucleotides adjacent to a double-stranded stem. Like RNase T, Rex1p participates in 5S and tRNA 3′ maturation and CCA turnover. However, while Rex1p and RNase T are both members of the DEDD superfamily of exonucleases, Rex1p lacks motifs that form a substrate-binding patch in RNase T (Zuo et al. 2007). Rex1p is also larger than an RNase T monomer (63 kDa versus ~24 kDa), with N- and C-terminal domains that are absent in the prokaryotic enzyme. Since Rex1p appears unique among S. cerevisiae exonucleases in being able to access pre-tRNA ends in Lhp1p-containing cells, these domains could contribute to high-affinity RNA binding. Significantly, while RNase T orthologs have not been detected outside γ-proteobacteria (Zuo and Deutscher 2001), likely Rex1p orthologs are present in the genomes of plants and mammals. Thus, the roles of Rex1p in pre-tRNA and 5S rRNA maturation and in generating TRAMP substrates may be widespread in eukaryotes.

Finally, the result that the end-matured intron-containing forms of pre-tRNATyr GUA and pre-tRNASer CGA in wild-type cells largely contain CC- rather than CCA suggests that Rex1p-mediated end turnover could contribute to nuclear retention of uncharged pre-tRNAs. Experiments in yeast have shown that although aminoacylation enhances tRNA export, it is not required (Azad et al. 2001). However, inactivation of the CCA-adding enzyme results in a rapid block in export, and the export receptor exportin-t/Los1p preferentially binds CCA-containing tRNAs (for review, see Hopper 2006). Since aminoacylated tRNAs are exported, Rex1p-mediated end turnover could function to retard export of those pre-tRNAs that fail to undergo aminoacylation. In this scenario, uncharged nuclear pre-tRNAs may undergo cycles of Rex1p-dependent end trimming and repair by the CCA-adding enzyme. Thus, at steady state those pre-tRNAs that are present in nuclei will be enriched for RNAs with incompletely mature ends.

Noncoding RNA quality control

Our experiments have expanded the list of TRAMP substrates and revealed that Rex1p contributes to the generation of at least some of these RNAs. Previously, the only known pre-tRNA substrate for the TRAMP-mediated quality-control pathway was a hypomethylated form of pre-tRNAMet i that is polyadenylated prior to end maturation (Kadaba et al. 2004). The finding that end-matured forms of eight different intron-containing pre-tRNAs accumulate in trf4Δ cells indicates that unspliced pre-tRNAs are also TRAMP targets. However, since our experiments detect only RNA that accumulates at steady state, we do not know at which stage the pre-tRNAs that accumulate in trf4Δ cells are actually targeted for degradation by TRAMP. Indeed, since aminoacylation will block polyadenylation, it is likely that TRAMP normally targets these RNAs at an earlier stage in their biogenesis.

Why should Rex1p trimming of pre-tRNA trailers result in a population of unspliced, end-matured pre-tRNAs that are TRAMP substrates? Since Lhp1p stabilizes the anticodon stem of some pre-tRNAs (Chakshusmathi et al. 2003), one possibility is that a fraction of intron-containing pre-tRNAs that fail to bind Lhp1p fold into alternative structures. These altered conformations may slow the rate of end maturation, CCA addition and/or aminoacylation, making them targets for TRAMP. In trf4Δ cells, these defective pre-tRNAs would not undergo polyadenylation and degradation, allowing end maturation and some aminoacylation, but they may be poorly recognized by either nuclear export receptors and/or the cytoplasmic pre-tRNA splicing machinery. Consistent with the trapping of RNA in incorrect helices, more intron-containing pre-tRNAs accumulate in trf4Δ cells during growth at 25°C than at higher temperatures (data not shown). An alternative but not exclusive possibility is that cleavage by the 3′ endonuclease is specific for correctly folded pre-tRNAs. In this case, the population of pre-tRNAs that undergo exonucleolytic maturation in wild-type cells may be enriched in pre-tRNAs with perturbed structures.

Importantly, the finding that the accumulation of truncated 5S and SRP RNAs in trf4Δ cells requires Rex1p indicates that Rex1p plays a general role in generating TRAMP substrates. Since 5S rRNA is normally generated by Rex1p trimming of the 3′-extended precursor (van Hoof et al. 2000), a likely scenario is that the truncated RNA is generated by the failure of Rex1p to stop at the correct mature end. For SRP RNA, the mature end of the RNA coincides with the termination site, consisting of 3–5 uridylates that allow Lhp1p binding (Felici et al. 1989; Yoo and Wolin 1994). One possibility is that nascent SRP RNAs that fail to bind Lhp1p undergo Rex1p-depending nibbling of the 3′ end, resulting in defective RNAs that are targeted for decay.

A puzzling aspect of our data is the result that less end-matured unspliced pre-tRNAs accumulate in trf4Δ lhp1Δ cells than trf4Δ cells (Figs. 1B, ,2A).2A). Since binding by La protects the 3′ ends of RNAs from exonucleases, it might have been expected that the levels of defective pre-tRNAs would increase in trf4Δ lhp1Δ cells. We have shown that Rex1p is the major 3′ to 5′ exonuclease that accesses pre-tRNA 3′ ends in wild-type cells, but that additional exonucleases, such as Rrp6p, access these RNAs in lhp1Δ cells. One possibility is that Rex1p-dependent maturation results in a larger population of pre-tRNAs that can be polyadenylated by TRAMP than does maturation by other nucleases. For example, if Rex1p is distributive, its dissociation from the trailer during maturation may allow TRAMP to access the RNA. In trf4Δ lhp1Δ cells, more processive exonucleases may access the RNA, resulting in a lower availability of free 3′ ends. An alternative possibility is that these other exonucleases can degrade defective pre-tRNAs independently of TRAMP. In support of this idea, the exosome subunit Rrp44p/Dis3p can degrade a hypomethylated pre-tRNA to completion in vitro, although its activity is stimulated by TRAMP (Schneider et al. 2007).

Taken together, our data indicate that Lhp1p, Rex1p, and possibly also TRAMP compete for access to nascent RNAs. Consistent with this model, overexpression of LHP1 suppresses degradation of hypomethylated pre-tRNAMet i (Anderson et al. 1998), while decay of this pre-tRNA is enhanced in lhp1Δ cells (Calvo et al. 1999). Since Lhp1p binding requires UUUOH (Wolin and Cedervall 2002), those nascent RNAs containing less than three terminal uridines will undergo exonucleolytic trimming and/or TRAMP-mediated decay. For RNAs that misfold into abnormal structures that slow the rate of end maturation, competition between Rex1p and Lhp1p may eventually result in a 3′ end with less than three uridines, allowing additional exonucleolytic nibbling and/or TRAMP polyadenylation. Although we do not know whether TRAMP normally targets the intron-containing pre-tRNAs during exonucleolytic maturation or following CCA addition, either possibility is consistent with the hypothesis that TRAMP targets RNAs that possess an accessible 3′ end (Reinisch and Wolin 2007). Accessible ends could be generated by the failure to bind Lhp1p, delays in end maturation or nuclear aminoacylation, the failure to bind export receptors, or the failure of nascent RNAs to assemble with their correct RNA-binding proteins.

MATERIALS AND METHODS

Media and strains

Media was prepared according to Sherman (1991). The strains used in this study are listed in Table 1. The rex1Δ, rex2Δ, and rex4Δ strains were generated by back-crossing strain YAV140 (a gift of A. van Hoof, University of Texas Health Science Center, Houston) once to CY4 and twice to CY3. Null alleles of REX3, TRF5, and RRP6 were constructed using PCR to replace most or all of the coding sequence with a selectable marker. For REX3, nucleotides 23–1120 were replaced with URA3, while for TRF5, the entire coding sequence was replaced with HIS3. For rrp6::kanr, nucleotides 118–1500 of the coding sequence were replaced with kan r (Longtine et al. 1998). The rna1-1 strain EE1b6 and the cca1-1 strain ts352 were gifts of A. Hopper (Ohio State University).

TABLE 1.
Yeast strains used in this study

Northern analyses

Yeast were grown at 25°C to OD600 between 0.2 and 0.5, and RNA was extracted with hot acid phenol as described (Ausubel et al. 1998). For Northern analyses, RNA was fractionated in 5% or 8% polyacrylamide/8.3 M urea gels and transferred to Hybond N (G.E. Healthcare) in 0.5× TBE buffer at 150 mA for 16 h. To examine aminoacylation, RNA was extracted at low pH, fractionated in 6.5% polyacrylamide, 8 M urea, 0.1 M NaOAc (pH 5.0) gels at 4°C (Varshney et al. 1991) and transferred as above. Filters were hybridized with [γ-32P]ATP-labeled oligonucleotides as described (Tarn et al. 1995). Oligonucleotides were:

  • pre-tRNASer CGA: 5′-TAGCCGAACTTTTTATTCCATTCGAG-3′;
  • tRNASer CGA: 5′-AGAGCCCAAGAGATTTCGAGTCTCTCGCCTT-3′;
  • tRNAArg CCG: 5′-ACTCGAACCCGGATCACAGCCACCGGAAGAATGCATGCTAACCATT-3′;
  • U4: 5′-AGGTATTCCAAAAATTCCCTAC-3′;
  • U6: 5′-CGAAATAAATCTCTTTGTAAAACGG-3′;
  • U3: 5′-TTCGGTTTCTCACTCTG-3′;
  • pre-tRNALeu UAG: 5′-GATATCAGAGATTTTAGAGGTTAAATCCACCT-3′;
  • tRNALeu UAG: 5′-AAGATATCAGAGCCTAAATCTGACG-3′;
  • pre-tRNALys UUU: 5′-ACATTTCGGTTAAAAGCCGAACG-3′;
  • tRNALys UUU: 5′-TGACATTTCGGTTAAAAGCCGAACGCTCTACCAAC-3′;
  • pre-tRNATyr GUA: 5′-CCGATCTCAAGATTTCGTAGTGATAAATTAC-3′;
  • tRNATyr GUA: 5′-GATCTCAAGATTTACAGTCTTGCG-3′;
  • pre-tRNAPro UGG: 5′-TGCTTTGTCTTCCTGTTTAATCAGGAAGTC-3′;
  • tRNAPro UGG: 5′-GGCCTCTCGCACCCAAAGCG-3′;
  • pre-tRNASer GCU: 5′-GCCTAATTGCTTTTCTGAGGAAA-3′;
  • tRNASer GCU: 5′-CCCAATGCCTTAGCAGGGCA-3′;
  • pre-tRNAPhe GAA: 5′-TCCAGATGACTTGACCGAAGT-3′;
  • tRNAPhe GAA: 5′-GACCTCCAGATCTTCAGTCTGGC-3′;
  • 5S rRNA: 5′-CCAGCTTAACTACAGTTGATCGGACGGGAA-3′; and
  • SRP RNA: 5′-TCAACGTATCCCATCCCAC-3′.

Immunoprecipitations

Cultures were grown in YPD at 25°C to OD600 = 0.5, washed twice with H2O, and resuspended in 600 μL of NET-2 (40 mM Tris-HCl at pH 7.5, 150 mM NaCl, 0.05% Nonidet P-40) and 0.25 mM phenylmethylsulfonyl fluoride (PMSF). After vortexing with glass beads, extracts were cleared by sedimentation for 5 min at 2000 rpm in a microcentrifuge followed by 100,000g for 20 min in a Beckman TLA100.2 rotor. Extracts were incubated with anti-Lhp1p antibodies (Yoo and Wolin 1994) that were pre-bound to Protein A Sepharose CL-4B (G.E. Healthcare). After 1 h at 4°C, the beads were washed six times with NET-2. RNA was extracted with phenol/chloroform/isoamyl alcohol (50:50:1) in the presence of 0.2% SDS, followed by chloroform/isoamyl alcohol (50:1) and precipitation with ethanol.

Oligonucleotide splint labeling

Total RNA (10 μg) from wild-type, rex1Δ, trf4Δ, and rna1-1 cells was mixed with 20 pmol each of the CCA and trailer-containing oligonucleotides in 40 mM Tris-HCl (pH 7.5), 50 mM NaCl, 10 mM MgCl2, 0.5 mM EDTA, and 1 mM DTT. After heating for 2 min to 85°C and cooling on ice for 30 min, dATP and dTTP were added to 100 mM each, and 5 units of Sequenase Version 2.0 (U.S. Biochemical) were added. After incubation for 1 h at 37°C, reactions were stopped by adding 200 μL of 0.2 mg/mL proteinase K, 50 mM Tris (pH 7.5), 50 mM NaCl, 5 mM EDTA, and 0.5% SDS and incubating for 30 min at 37°C. Following ethanol precipitation, samples were fractionated in 8% polyacrylamide, 8.3 M urea gels. Oligonucleotides were:

  • tRNATyr GUA trailer: 5′-GTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTAA/TATCTCCCGGGGGCGAGTCGAA-3′;
  • tRNATyr GUA CCA: 5′-GAAAAAAAAAAAAAAAAAAAAAAAAATGGTCTCCCGGGGGCGAGTCGAA-3′;
  • tRNASer CGA trailer: 5′-GTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTAAACGACACCAGCAGGATTTGAAC-3′; and
  • tRNASer CGA CCA: GAAAAAAAAAAAAAAAAAAAAAAAAATGGCGACACCAGCAGGATTTGAAC-3′.

ACKNOWLEDGMENTS

We thank Ambro van Hoof, Roy Parker, and Anita Hopper for gifts of strains and Andrei Alexandrov, Soyeong Sim, and Elisabeth Wurtmann for comments on the manuscript. This work was supported by National Institutes of Health Grant R01-GM48410.

Footnotes

Article published online ahead of print. Article and publication date are at http://www.rnajournal.org/cgi/doi/10.1261/rna.1050408.

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