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Three conserved members of the RNase D family have unique and overlapping functions in the processing of 5S, 5.8S, U4, U5, RNase MRP and RNase P RNAs in yeast

2000; Springer Nature; Volume: 19; Issue: 6 Linguagem: Inglês

10.1093/emboj/19.6.1357

1460-2075

Ambro van Hoof,

RNA and protein synthesis mechanisms

Article15 March 2000free access Three conserved members of the RNase D family have unique and overlapping functions in the processing of 5S, 5.8S, U4, U5, RNase MRP and RNase P RNAs in yeast Ambro van Hoof Corresponding Author Ambro van Hoof Department of Molecular and Cellular Biology, Howard Hughes Medical Institute, University of Arizona, Tucson, AZ, 85721 USA Search for more papers by this author Pascal Lennertz Pascal Lennertz Department of Molecular and Cellular Biology, Howard Hughes Medical Institute, University of Arizona, Tucson, AZ, 85721 USA Search for more papers by this author Roy Parker Corresponding Author Roy Parker Department of Molecular and Cellular Biology, Howard Hughes Medical Institute, University of Arizona, Tucson, AZ, 85721 USA Search for more papers by this author Ambro van Hoof Corresponding Author Ambro van Hoof Department of Molecular and Cellular Biology, Howard Hughes Medical Institute, University of Arizona, Tucson, AZ, 85721 USA Search for more papers by this author Pascal Lennertz Pascal Lennertz Department of Molecular and Cellular Biology, Howard Hughes Medical Institute, University of Arizona, Tucson, AZ, 85721 USA Search for more papers by this author Roy Parker Corresponding Author Roy Parker Department of Molecular and Cellular Biology, Howard Hughes Medical Institute, University of Arizona, Tucson, AZ, 85721 USA Search for more papers by this author Author Information Ambro van Hoof 1, Pascal Lennertz1 and Roy Parker 1 1Department of Molecular and Cellular Biology, Howard Hughes Medical Institute, University of Arizona, Tucson, AZ, 85721 USA *Corresponding authors. E-mail: [email protected] or E-mail: [email protected] The EMBO Journal (2000)19:1357-1365https://doi.org/10.1093/emboj/19.6.1357 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The biogenesis of a number of RNA species in eukaryotic cells requires 3′ processing. To determine the enzymes responsible for these trimming events, we created yeast strains lacking specific 3′ to 5′ exonucleases. In this work, we describe the analysis of three members of the RNase D family of exonucleases (Rex1p, Rex2p and Rex3p). This work led to three important conclusions. First, each of these exonucleases is required for the processing of distinct RNAs. Specifically, Rex1p, Rex2p and Rex3p are required for 5S rRNA, U4 snRNA and MRP RNA trimming, respectively. Secondly, some 3′ exonucleases are redundant with other exonucleases. Specifically, Rex1p and Rex2p function redundantly in 5.8S rRNA maturation, Rex1p, Rex2p and Rex3p are redundant for the processing of U5 snRNA and RNase P RNA, and Rex1p and the exonuclease Rrp6p have an unknown redundant essential function. Thirdly, the demonstration that the Rex proteins can affect reactions that have been attributed previously to the exosome complex indicates that an apparently simple processing step can be surprisingly complex with multiple exonucleases working sequentially in the same pathway. Introduction The production of a wide variety of stable RNA species in eukaryotic cells requires specific 3′ exonucleolytic trimming reactions. This is in sharp contrast to how the end of most proteins is generated, where most of the C-termini of mature proteins correspond to the site of termination of synthesis. One possible explanation for the widespread use of 3′ RNA processing is that it allows for flexibility in the sequence of the 3′ end. This is due to the fact that the sequences that specify the 3′ end of the primary transcript restrict the possible sequences near its 3′ end. For example, the transcription termination signal for RNA polymerase III is a run of Us, such that the primary transcript necessarily ends in a run of Us. Similarly, the primary transcript of RNA polymerase II ends in a poly(A) tail. In addition, the information to cleave and polyadenylate is mostly located near the 3′ end of the primary transcript, putting further restrictions on the sequence of the primary transcript. Generating the 3′ end of stable RNA species by a processing reaction clearly allows for more flexibility in the sequence of the 3′ end of the mature transcripts. A key step in understanding the importance of RNA 3′-processing reactions is the identification and analysis of the 3′ exoribonucleases that process RNAs. Examination of the yeast genome sequence predicted 17 3′ to 5′ exonucleases (Mian, 1996; Moser et al., 1997). In addition, at least one yeast protein (Rrp4p) not predicted to have 3′ exonuclease activity has been found to have such an activity (Mitchell et al., 1997). The presence of many 3′ exonucleases raises several questions, such as whether individual exonucleases are required for the processing of specific RNA species, and if so, what the specific roles of each exonuclease are. Alternatively, most of the exonucleases could be redundant with each other, with any one nuclease able to process a specific RNA species. Some information is available on the function of selected yeast exonucleases. Nine of the (putative) 3′ exonucleases (Rrp4p, Rrp6p, Rrp41p, Rrp42p, Rrp43p, Rrp44p, Rrp45p, Rrp46p and Mtr3p) are present in one protein complex named the exosome. This complex is found in both the cytoplasm and the nucleus, and is thought to function in the processing of rRNA, snRNAs and snoRNAs and in the degradation of mRNAs and the external transcribed spacer of the rRNA. (Mitchell et al., 1997; Jacobs Anderson and Parker, 1998; Allmang et al., 1999a,b; van Hoof et al., 2000). A second class of interesting 3′ exonucleases consists of five related yeast proteins that are part of a large family of proteins that includes known 3′ exoribonucleases [i.e. RNase D, RNase T and oligoribonuclease from Escherichia coli, Rrp6p from yeast and PARN from human and Xenopus (Moser et al., 1997; Korner et al., 1998)]. One of these five yeast proteins, Pan2p, has been shown to play a role in initial shortening of the poly(A) tails of mRNA (Boeck et al., 1996; Brown and Sachs, 1998). We have named the other four proteins Rex1p [RNA exonuclease 1; open reading frame (ORF) yGR276], Rex2p (ORF yLR059), Rex3p (ORF yLR107) and Rex4p (ORF yOL080). REX2 has recently been identified as YNT20 (Hanekamp and Thorsness, 1999), a suppressor of yme1-mediated escape of DNA from the mitochondrion. It was also shown that epitope-tagged Ynt20p when overexpressed sediments in a 10 min 10 000 g centrifugation step (Hanekamp and Thorsness, 1999). This, together with a cold-sensitive respiratory growth defect, was interpreted as evidence for a mitochondrial localization of Rex2p. REX1 has recently been identified as RNH70, a 70 kDa protein that copurifies with RNase H activity. However, Rex1p/Rnh70p does not show any sequence similarity to known RNase H proteins and rex1/rnh70 mutants do not show reduced RNase H activity, or the expected phenotype for an RNase H mutant (Frank et al., 1999; Qiu et al., 1999). It is therefore not clear whether Rex1p/Rnh70p indeed functions as an RNase H in vivo, and if so, whether it has exonuclease functions in addition to the RNase H function. The Rex1p, Rex2p, Rex3p, Rex4p and Pan2p proteins are well conserved, with at least four of them having homologs in the human genome. The proteins encoded by the uncharacterized human ORFs BAA31685, AAC31668 and CAB53690 are homologs of Pan2p, Rex1p and Rex2p, respectively. Rex4p homologs have been studied to some extent in human and Xenopus. The human gene is named either ISG20 or HEM45, and encodes a protein whose expression is induced by interferon or estrogen and localizes to the nucleus (Gongora et al., 1997; Pentecost, 1998). The Xenopus homolog (XPMC2) is also a nuclear protein that, by an unknown mechanism, can rescue a cell cycle defect when expressed in a mutant fission yeast (Su and Maller, 1995). Given the conservation of this family of 3′ to 5′ exonucleases, we analyzed their possible role in RNA-processing reactions. Here we report specific RNA-processing defects for three of these conserved proteins. In addition, this work clarifies the role of the exosome in some RNA-processing reactions. Results To identify what role the Rex1p, Rex2p Rex3p and Rex4p putative exonucleases play in the cell we created null mutations in the REX1, REX2, REX3 and REX4 genes by precise deletion of each of the coding regions from the yeast genome. Each of these deletion mutants is alive, indicating that these proteins are not required for viability. In addition, we did not observe any growth defects on a variety of carbon sources and at a variety of temperatures in any of these mutants (data not shown). In our strains, none of the mutations in rex genes results in a cold-sensitive growth defect on glycerol. This is different from previous reports of rex2/ynt20 mutations (Hanekamp and Thorsness, 1999). Since we anticipated that there might be functional overlap between some of these proteins, we also carried out similar analyses in strains deleted for various combinations of these genes, and also failed to find an obvious growth defect in any of these multiple mutants (including the rex1Δ, rex2Δ, rex3Δ, rex4Δ, pan2Δ pentuple mutant; data not shown). In order to determine if the Rex proteins were required for any 3′ trimming reactions we isolated RNA from each mutant and analyzed it by Northern blotting for aberrant processing of various RNA species. While each of the rex1Δ, rex2Δ and rex3Δ mutants does show specific RNA-processing defects (see below), none of the mutants tested showed an obvious defect in a number of other RNA species, including several tRNAs, 7S RNA, U6 snRNA and several snoRNAs (data not shown). These results serve as important controls and indicate that the defects described below reflect specific roles for these genes. As additional negative controls, we analyzed strains deleted for the putative exonuclease genes SSD1, yDR514 and yCL036. These latter mutants, as well as rex4Δ, did not have any obvious growth defects, nor did they show defects in the processing of any of the RNA species tested, and thus serve as additional negative controls (data not shown). Rex1p is required for 5S and tRNA-Arg3 maturation Strains deleted for REX1, but not REX2 or REX3, accumulate 5S rRNA that is longer than the corresponding RNA in wild type by ∼3 nt (Figure 1A). 5S rRNA is transcribed by RNA polymerase III as a precursor with 3′ extensions. Therefore, we hypothesized that Rex1p was required for the removal of these 3′ extensions. To test this possibility, we checked whether the longer form of 5S rRNA indeed carried 3′ extensions by cleaving the RNA with RNase H and probing for the 3′ cleavage product. As shown in Figure 1B, rex1Δ strains indeed accumulated 3′ extended forms of 5S rRNA. We interpret these observations to indicate that Rex1p is required for the proper maturation of the 3′ end of the 5S RNA (see Discussion). Figure 1.rex1Δ results in defects in 5S rRNA and tRNA-Arg3 processing, but not tRNA-Ser5. The strains indicated were grown to early- to mid-log phase in YPD at 30°C. RNA was extracted and analyzed by Northern blotting. Numbers to the left of each panel indicate the position of DNA molecular weight markers. (A) A Northern blot was probed for 5S rRNA. The migration of mature 5S rRNA from wild-type strains is indicated. (B) 5S rRNA was cleaved with oRP921 and RNase H, or treated with RNase H in the absence of any oligonucleotide before electrophoresis. The Northern blot was probed for 5S rRNA. The positions of mature 5S rRNA and the 3′ RNase H cleavage fragment are indicated. (C) A Northern blot was probed for 3′ extended forms of tRNA-Arg3. The migrations of a dicistronic precursor, as well as the monomeric processing intermediate with 5′ and 3′ extensions, are indicated. Also indicated is the migration of mature tRNA-Arg3, which does not hybridize to this probe. The faint band visible between 110 and 147 nt is a remaining signal from a previous probing for 5S rRNA that did not completely strip off. (D) The same Northern blot as in (C) was reprobed for intron-containing precursors to tRNA-Ser5. The positions of unspliced precursor, with and without 5′ and 3′ extensions, are indicated. Download figure Download PowerPoint Strains lacking Rex1p were also defective in the processing of tRNA-Arg3. tRNA-Arg3 is encoded by four dicistronic genes and seven monocistronic genes (Hani and Feldmann, 1998). The precursor transcript from the dicistronic genes is processed into two monomeric intermediates. The 5′ cistron is then further processed, possibly by a 3′ exonuclease, to yield tRNA-Arg3 (Schmidt et al., 1980; Engelke et al., 1985). Figure 1C shows the altered pattern of accumulation of 3′ extended tRNA-Arg3 precursors in rex1Δ strains. This blot was probed with an oligonucleotide probe that is specific for 3′ extended forms of tRNA-Arg3 derived from the dicistronic tRNA-Arg3-Asp gene. We interpret these observations to indicate that Rex1p is required for the proper maturation of the 5′ cistron of this tRNA dicistronic unit. No gross differences were seen when similar blots were probed for precursors to two other tRNAs (tRNA-Leu3 and tRNA-Ser5; Figure 1D and data not shown). The observation that Rex1p is not required for the processing of other tRNAs is not surprising. All other tRNA genes are transcribed as monomeric RNAs (Hani and Feldmann, 1998). The 3′ ends of these monomeric tRNAs are thought to be processed by endonucleolytic cleavage in an Lhp1p-dependent manner (Yoo and Wolin, 1997). Lhp1p is an RNA-binding protein associated with the 3′ end of nascent RNA polymerase III transcripts. The 5′ cistron of the dimeric transcript does not contain an RNA polymerase III transcription termination site and therefore is probably not bound by Lhp1p. Thus, this 5′ cistron can not be processed by an Lhp1p-dependent endonuclease. The phenotypes we described above for the rex1 deletion are similar to the phenotypes previously described for the rna82-1 mutation (Piper et al., 1983; Piper and Straby, 1989). Since the RNA82 gene has not been cloned it could either be a second gene required for the same processing steps, or RNA82 and REX1 could be the same gene. We tested whether rex1Δ and rna82-1 were in the same complementation group by crossing the two mutant strains to each other, and crossing each of them to a wild-type strain. Northern blot analyses revealed that indeed the 5S rRNA- and tRNA-processing defects were identical in the two mutants, and that they did fall in the same complementation group (Figure 2 and data not shown). To confirm that REX1 and RNA82 were the same gene we sequenced the REX1 gene from the rna82-1 strain, and found that this gene contained a mutation changing Trp433 to a stop codon. We thus conclude that REX1 and RNA82 are the same gene. Figure 2.rex1Δ and rna82-1 fail to complement each other. The strains indicated were grown to early- to mid-log phase in YPD at 30°C and analyzed by Northern blotting and probing for 5S rRNA. Numbers to the left indicate the position of DNA molecular weight markers. Download figure Download PowerPoint Rex2p is required for proper U4 snRNA maturation Strains deleted for REX2, but not REX1 or REX3, accumulate U4 snRNAs that were ∼1–4 nucleotides (nt) longer than the 160 nt RNA in wild type (Figure 3A). Moreover, cleavage of U4 snRNA with an oligonucleotide and probing for the 3′ fragment showed that the increase in size was due to a difference in the 3′ end of these molecules (Figure 3B). We interpret these observations to indicate that Rex2p is required for the final trimming of the 3′ end of U4 snRNA. Figure 3.rex2Δ results in a defect in U4 snRNA processing. The strains indicated were grown to early- to mid-log phase in YPD at 30°C. RNA was extracted and analyzed by Northern blotting. Numbers to the left of (B) and (C) indicate the position of DNA molecular weight markers. (A) A Northern blot was probed for U4 snRNA. The migration of mature U4 snRNA from wild-type strains is indicated. (B) U4 snRNA was cleaved with oRP756 and RNase H, or treated with RNase H in the absence of any oligonucleotide before electrophoresis. The Northern blot was probed for U4 snRNA. The positions of mature U4 snRNA and the 3′ RNase H cleavage fragment are indicated. (C) A dark (upper panel) and light (lower panel) exposure of the same Northern blot probed for U4 snRNA is shown. The migration of mature U4 snRNA from wild-type strains is indicated. Download figure Download PowerPoint The 3′ trimming of the U4 transcript has been attributed previously to the exosome complex of 3′ exonucleases (Allmang et al., 1999b; van Hoof et al., 2000). Previous analyses have shown that U4 snRNA is initially transcribed as a species of >300 nt (and likely to be >500 nt). This precursor is cleaved by Rnt1p (an endonuclease) to a 295 nt product that contains U4 with a 135 nt 3′ extension. Mutants in RRP6 (and other exosome mutants) are defective in the processing of this Rnt1p cleavage product (Figure 3C; Allmang et al., 1999b; van Hoof et al., 2000). In contrast, the rex2Δ mutant showed a defect in removal of the last few nucleotides of the 3′ extension. This suggests that Rex2p acts on the product of trimming by the exosome. Interestingly, analysis of the rrp6Δ, rex2Δ double mutant showed that the rex2Δ phenotype was exacerbated. The rex2Δ, rrp6Δ double mutant contained little if any U4 snRNA of the normal size (Figure 3C, lower panel) indicating that in a rex2Δ mutant, Rrp6p (or perhaps the exosome) can partially take over the Rex2p role. These results suggest that the processing of the U4 snRNA either involves parallel pathways in which different exonucleases perform the trimming reactions or that the processing of this RNA requires the sequential action of various different exonucleases (see Discussion). Rex3p is required for proper MRP RNA maturation Strains deleted for REX3, but not REX1 or REX2, accumulated RNase MRP RNAs that were ∼7 nt longer than the corresponding RNA in wild type (Figure 4A). Moreover, cleavage of MRP RNA with an oligonucleotide and probing for the 3′ half showed that the increase in size was due to a difference in the 3′ end of these molecules (Figure 4B). We therefore conclude that Rex3p plays a role in 3′ end formation of the RNA subunit of RNase MRP. Figure 4.rex3Δ results in a defect in the processing of the RNA subunit of RNase MRP. The strains indicated were grown to early- to mid-log phase in YPD at 30°C. RNA was extracted and analyzed by Northern blotting. Numbers to the left of each panel indicate the position of DNA molecular weight markers. (A) A Northern blot was probed for the MRP RNA. The migration of mature MRP RNA from wild-type strains is indicated. (B) The MRP RNA was cleaved with oRP920 and RNase H, or treated with RNase H in the absence of any oligonucleotide before electrophoresis. The Northern blot was probed for MRP RNA. The positions of mature MRP RNA and the 3′ RNase H cleavage fragment are indicated. Download figure Download PowerPoint Some combinations of 3′ exonucleases have redundant roles in the maturation of specific RNAs We anticipated that some 3′ trimming reactions could be performed by multiple 3′ to 5′ exonucleases. Given this, we created and analyzed the phenotypes of a variety of multiple mutants that revealed a number of overlapping functions for these putative 3′ to 5′ exonucleases. Rex1p and Rex2p are functionally redundant in the maturation of 5.8S rRNA. Analyses of various RNA-processing events in a rex1Δ, rex2Δ double mutant revealed that these two proteins have a redundant role in the processing of 5.8S rRNA. While each single mutant accumulated normal levels of 3′ extended precursors to 5.8S rRNA, the double mutant accumulated a species that we estimate to be ∼8 nt longer than the mature 5.8S rRNA (Figure 5A). This species is the correct size to correspond to the 6S pre-rRNA previously described in wild-type yeast strains (Mitchell et al., 1996). Elevated levels of 6S rRNA were not seen in any of the three single rexΔ strains, the rex1Δ, rex3Δ or rex2Δ, rex3Δ double mutants (Figure 5A and data not shown). Interestingly, the accumulation of the 6S rRNA species was higher in the rex1Δ, rex2Δ, rex3Δ triple mutant strain than in the rex1Δ, rex2Δ double mutant strain (Figure 5A), suggesting that Rex3p can also process 6S rRNA, albeit at a slower rate (see Discussion). Figure 5.Rex1p and Rex2p are redundant for 5.8S rRNA processing. The strains indicated were grown to early- to mid-log phase in YPD at 30°C. RNA was extracted and analyzed by Northern blotting. (A) A Northern blot was probed for 3′ extended forms of 5.8S rRNA. The positions of 7S pre-rRNA and 6S pre-rRNA are indicated. Numbers to the left of the panel indicate the position of DNA molecular weight markers. (B) The same Northern blot was reprobed with a probe for the mature 5.8S rRNA. The positions of both L and S isoforms of mature 5.8S rRNA and 6S pre-rRNA are indicated. These L and S isoforms differ from each other at their 5′ end. (C) A Northern blot was probed for 3′ extended forms of 5.8S rRNA. The positions of 7S pre-rRNA, 5.8S+30 pre-rRNA and 6S pre-rRNA are indicated. Download figure Download PowerPoint To test to what extent rex mutants accumulate 3′ extended 5.8S rRNA we reprobed a blot containing RNA from the rex1Δ, rex2Δ, rex3Δ triple mutant with a probe for the mature 5.8S rRNA. This showed (Figure 5B) that, although most of the 5.8S rRNA in this strain is of the correct size, the rex1Δ, rex2Δ, rex3Δ triple mutant does accumulate a few percent of its 5.8S rRNA as a 6S species (see Discussion). Together these results suggest that Rex1p and Rex2p function redundantly in the 3′ end processing of 5.8S rRNA, but that Rex3p and an unidentified nuclease can to some extent substitute for Rex1p and Rex2p. The 3′ trimming of pre-5.8S rRNA has been attributed previously to the exosome complex of 3′ exonucleases. Previous analyses indicate that the 156 nt 5.8S rRNA is initially co-transcribed with 18S and 25S rRNA as one large precursor. This precursor is processed by a combination of endonucleolytic cleavages and 5′ exonucleases to a 7S species of ∼300 nt. This 7S species is then thought to be processed by the exosome (Mitchell et al., 1997). Consequently, rrp6Δ mutants accumulate 5.8S rRNA precursors with ∼30 nt extensions (Briggs et al., 1998). Figure 5C shows that the 3′ extensions seen in rrp6Δ are longer than the ones seen in the rex1Δ, rex2Δ double mutant or the rex1Δ, rex2Δ, rex3Δ triple mutant. This suggests that the exosome acts on the 7S intermediate, and processes it to a 6S intermediate, which is subsequently processed by either Rex1p or Rex2p to yield ultimately the mature 5.8S rRNA. In order to test the relationship between processing of 5.8S pre-rRNA by Rex1p, Rex2p and Rrp6p we attempted to create a strain lacking all three of these non-essential genes. Surprisingly, analysis of 21 tetrads from a cross between a rex1Δ strain and an rrp6Δ strain only yielded 77% viable spores, and no rex1Δ, rrp6Δ double mutants were recovered. In other crosses spore viability was 90–100% and all other combinations of double mutants of rex1Δ, rex2Δ, rex3Δ and rrp6Δ were recovered at the expected frequency in these other crosses. This indicates that rex1Δ is synthetically lethal with rrp6Δ (at least during spore germination), and therefore suggests that the Rex1p and Rrp6p proteins have a redundant role in the processing of one or more RNA species. The identity of this redundant role is not clear. One candidate is the processing of 5.8S rRNA precursors, since both Rex1p and Rrp6p are involved in this process. However, Rex2p can substitute for Rex1p in 5.8S processing, but apparently can not efficiently substitute for the Rex1p/Rrp6p redundant function. Rex1p, Rex2p and Rex3p are functionally redundant in the maturation of U5L snRNA. Analyses of the rex1Δ, rex2Δ, rex3Δ triple mutant revealed an additional role for these proteins in the processing of U5 snRNA. Wild-type yeast accumulates two forms of U5 snRNA, named U5S snRNA and U5L snRNA. U5L snRNA has been shown to be a distinct 3′ extended form of mature U5 snRNA, and is not a precursor to U5S snRNA (Chanfreau et al., 1997). The rex1Δ, rex2Δ, rex3Δ triple mutant accumulated drastically reduced levels of U5L snRNA (Figure 6A). All three single rexΔ strains, and all three possible double mutant strains accumulate normal levels of U5L snRNA (Figure 6A and data not shown), indicating that Rex1p, Rex2p and Rex3p have a redundant role in the processing of U5L snRNA. In the absence of this processing event U5L snRNA precursors are apparently degraded, or processed to U5S. This is similar to the proposed degradation of precursors of U5L snRNA and snR44, when their processing is blocked by rnt1 and exosome mutations, respectively (Chanfreau et al., 1997; van Hoof et al., 2000). Figure 6.Rex1p, Rex2p and Rex3p are redundant for the processing of U5 snRNA. The strains indicated were grown to early- to mid-log phase in YPD at 30°C. RNA was extracted and analyzed by Northern blotting. Numbers to the left of each panel indicate the position of DNA molecular weight markers. (A) A Northern blot was probed for the U5 snRNA. The migration of mature U5S snRNA and U5L snRNA from wild-type strains is indicated. (B) The U5 snRNA was cleaved with oRP757 and RNase H, or treated with RNase H in the absence of any oligonucleotide before electrophoresis. The Northern blot was probed for U5 snRNA. The migration of mature U5S snRNA and U5L snRNA from wild-type strains and the 3′ RNase H cleavage fragment is indicated. (C) A Northern blot was probed for the U5 snRNA. The migration of mature U5S snRNA and U5L snRNA from wild-type strains is indicated. Download figure Download PowerPoint The rex1Δ, rex2Δ, rex3Δ triple mutant also accumulated slightly larger forms of U5S snRNA (Figure 6A). These larger forms were shown to be 3′ extended by RNase H cleavage and probing for the 3′ fragment (Figure 6B), and were also seen to a lesser extent in the rex2Δ single mutant. We conclude that Rex2p is required for normal U5S snRNA processing, but that Rex1p and Rex3p can substitute reasonably effectively for Rex2p. The 3′ trimming of this RNA has been attributed previously to the exosome complex of 3′ exonucleases (Allmang et al., 1999b). Comparison of the defects seen in U5L snRNA processing in rrp6Δ mutants with those seen in the rex1Δ, rex2Δ, rex3Δ triple mutants revealed that the rex triple phenotype was much more severe than the rrp6Δ phenotype (Figure 6C). We therefore conclude that the Rex proteins are primarily responsible for the 3′ processing of the U5L snRNA. The triple mutant may still accumulate low levels of U5L snRNA, suggesting that there is still another exonuclease that can process U5L snRNA precursors. We propose that this other exonuclease may be the exosome. Unfortunately we could not test this directly, since the rex1Δ, rex2Δ, rex3Δ, rrp6Δ quadruple mutant is inviable (data not shown), probably because of the synthetic lethality of rex1Δ and rrp6Δ described above. Rex1p, Rex2p and Rex3p are functionally redundant in the maturation of the RNA subunit of RNase P. The rex1Δ, rex2Δ, rex3Δ triple mutant showed a second defect in the processing of the RNA subunit of RNase P. The rex1Δ, rex2Δ, rex3Δ triple mutant accumulated a larger form of this RNA that was not present in any of the single or double mutants (Figure 7A and data not shown). This larger form was shown to be 3′ extended by probing a Northern blot with an oligonucleotide probe specific for 3′ extended forms of the RNA subunit of RNase P (Figure 7B). Wild-type yeast also accumulates two larger forms of RNase P, which are 5′ and 3′ extended and have been proposed to be precursors (Lee et al., 1991). The largest form of this putative precursor is also slightly larger in the rex1Δ, rex2Δ, rex3Δ triple mutant (Figure 7A). We interpret these results to suggest that the Rex1p, Rex2p and Rex3p proteins can all function redundantly to complete the proper maturation of the RNA subunit of RNase P. Figure 7.Rex1p, Rex2p and Rex3p are redundant for the processing of the RNA subunit of RNase P. The strains indicated were grown to early- to mid-log phase in YPD at 30°C. RNA was extracted and analyzed by Northern blotting. Numbers to the left of each panel indicate the position of DNA molecular weight markers. (A) A Northern blot was probed for the RNase P RNA. The migrations of mature RNase P RNA, a species with 5′ and 3′ extensions from wild-type strains and a 3′ extended species in the rex1Δ, rex2Δ, rex3Δ mutant are indicated. (B) A Northern blot was probed for 3′ extended forms of the RNA subunit of RNase P. Download figure Download PowerPoint Discussion The Rex1, Rex2 and Rex3 proteins are required for proper 3′ end maturation of several stable RNAs Our analyses identify the Rex proteins as being involved in a variety of RNA-processing reactions. The central observation is that strains lacking one or more of these proteins show the accumulation of 3′ extended forms of several RNAs. By this analysis, Rex1p is required for 5S rRNA and tRNA-Arg3 trimming, Rex2p is required for U4 snRNA trimming and Rex3p is required for trimming of the RNA subunit of RNase MRP. In addition, Rex1p and Rex2p function redundantly in 5.8S rRNA maturation, and Rex1p, Rex2p and Rex3p are redundant for the processing of U5 snRNA and the RNA subunit of RNase P. The identification of the Rex protein functions in RNA processing adds to