Joanna Kufel

FUNCTIONS OF THE EXOSOME IN RRNA, SNORNA AND SNRNA SYNTHESIS

The yeast nuclear exosome contains multiple 3′→5′ exoribonucleases, raising the question of why so many activities are present in the complex. All components are required during the 3′ processing of the 5.8S rRNA, together with the putative RNA helicase Dob1p/Mtr4p. During this processing three distinct steps can be resolved, and hand‐over between different exonucleases appears to occur at least twice. 3′ processing of snoRNAs (small nucleolar RNAs) that are excised from polycistronic precursors or from mRNA introns is also a multi‐step process that involves the exosome, with final trimming specifically dependent on the Rrp6p component. The spliceosomal U4 snRNA (small nuclear RNA) is synthesized from a 3′ extended precursor that is cleaved by Rnt1p at sites 135 and 169 nt downstream of the mature 3′ end. This cleavage is followed by 3′→5′ processing of the pre‐snRNA involving the exosome complex and Dob1p. The exosome, together with Rnt1p, also participates in the 3′ processing of the U1 and U5 snRNAs. We conclude that the exosome is involved in the processing of many RNA substrates and that different components can have distinct functions.

Precursors to the U3 Small Nucleolar RNA Lack Small Nucleolar RNP Proteins but Are Stabilized by La Binding

ABSTRACT Almost all small eukaryotic RNAs are processed from transiently stabilized 3′-extended forms. A key question is how and why such intermediates are stabilized and how they can then be processed to the mature RNA. Here we report that yeast U3 is also processed from a 3′-extended precursor. The major 3′-extended forms of U3 (U3-3′I and -II) lack the cap trimethylation present in mature U3 and are not associated with small nucleolar RNP (snoRNP) proteins that bind mature U3, i.e., Nop1p, Nop56p, and Nop58p. Depletion of Nop58p leads to the loss of mature U3 but increases the level of U3-3′I and -II, indicating a requirement for the snoRNP proteins for final maturation. Pre-U3 is cleaved by the endonuclease Rnt1p, but U3-3′I and -II do not extend to the Rnt1p cleavage sites. Rather, they terminate at poly(U) tracts, suggesting that they might be bound by Lhp1p (the yeast homologue of La). Immunoprecipitation of Lhp1p fused to Staphylococcus aureus protein A resulted in coprecipitation of both U3-3′I and -II. Deletion of LHP1, which is nonessential, led to the loss of U3-3′I and -II. We conclude that pre-U3 is cleaved by Rnt1p, followed by exonuclease digestion to U3-3′I and -II. These species are stabilized against continued degradation by binding of Lhp1p. Displacement of Lhp1p by binding of the snoRNP proteins allows final maturation, which involves the exosome complex of 3′→5′ exonucleases.

Polyadenylation Linked to Transcription Termination Directs the Processing of snoRNA Precursors in Yeast

Summary Transcription termination by RNA polymerase II is coupled to transcript 3′ end formation. A large cleavage and polyadenylation complex containing the major poly(A) polymerase Pap1 produces mRNA 3′ ends, whereas those of nonpolyadenylated snoRNAs in yeast are formed either by endonucleolytic cleavage or by termination, followed by trimming by the nuclear exosome. We show that synthesis of independently transcribed snoRNAs involves default polyadenylation of two classes of precursors derived from termination at a main Nrd1/Nab3-dependent site or a “fail-safe” mRNA-like signal. Poly(A) tails are added by Pap1 to both forms, whereas the alternative poly(A) polymerase Tfr4 adenylates major precursors and processing intermediates to facilitate further polyadenylation by Pap1 and maturation by the exosome/Rrp6. A more important role of Trf4/TRAMP, however, is to enhance Nrd1 association with snoRNA genes. We propose a model in which polyadenylation of pre-snoRNAs is a key event linking their transcription termination, 3′ end processing, and degradation.

Genetic and Physical Interactions Involving the Yeast Nuclear Cap-Binding Complex

ABSTRACT Yeast strains lacking the yeast nuclear cap-binding complex (yCBC) are viable, although impaired in growth. We have taken advantage of this observation to carry out a genetic screen for components that show synthetic lethality (SL) with a cbp20-Δcbp80-Δ double mutation. One set of SL interactions was due to mutations that were complemented by components of U1 small nuclear RNP (snRNP) and the yeast splicing commitment complex. These interactions confirm the role of yCBC in commitment complex formation. Physical interaction of yCBC with the commitment complex components Mud10p and Mud2p, which may directly mediate yCBC function, was demonstrated. Unexpectedly, we identified multiple SL mutations that were complemented by Cbf5p and Nop58p. These are components of the two major classes of yeast small nucleolar RNPs, which function in the maturation of rRNA precursors. Mutants lacking yCBC were found to be defective in rRNA processing. Analysis of the yCBC deletion phenotype suggests that this is likely to be due to a defect in the splicing of a subset of ribosomal protein mRNA precursors.

Microarray detection of novel nuclear RNA substrates for the exosome.

Microarray analyses were performed on yeast strains mutant for the nuclear‐specific exosome components Rrp6p and Rrp47p/Lrp1p or the core component Rrp41p/Ski6p, at permissive temperature and following transfer to 37 °C. 339 mRNAs showed clearly altered expression levels, with an unexpectedly high degree of heterogeneity in the different exosome mutants. In contrast, no clear alterations were seen in strains lacking the cytoplasmic exosome component Ski7p. 27 mRNAs that were overexpressed in each strain defective in the nuclear exosome are good candidates for regulation by nuclear turnover. These included the mRNA for the autoregulated RNA‐binding protein Nrd1p. Northern and primer extension analyses confirmed the elevated NRD1 mRNA levels in exosome mutants, and revealed the accumulation of truncated 5′ fragments of the mRNA. These contain a predicted Nrd1p‐binding site, potentially sequestering the protein and disrupting its autoregulation. Several genes located immediately downstream of independently transcribed snoRNA genes were overexpressed in exosome mutants, presumably due to stabilization of the products of transcription termination read‐through. Further analyses indicated that many snoRNA and snRNA genes are inefficiently terminated, but read‐through transcripts into downstream ORFs are normally rapidly degraded by the exosome. Copyright

Arabidopsis thaliana XRN2 is required for primary cleavage in the pre-ribosomal RNA

Three Rat1/Xrn2 homologues exist in Arabidopsis thaliana: nuclear AtXRN2 and AtXRN3, and cytoplasmic AtXRN4. The latter has a role in degrading 3′ products of miRNA-mediated mRNA cleavage, whereas all three proteins act as endogenous post-transcriptional gene silencing suppressors. Here we show that, similar to yeast nuclear Rat1, AtXRN2 has a role in ribosomal RNA processing. The lack of AtXRN2, however, does not result in defective formation of rRNA 5′-ends but inhibits endonucleolytic cleavage at the primary site P in the pre-rRNA resulting in the accumulation of the 35S* precursor. This does not lead to a decrease in mature rRNAs, as additional cleavages occur downstream of site P. Supplementing a P-site cleavage-deficient xrn2 plant extract with the recombinant protein restores processing activity, indicating direct participation of AtXRN2 in this process. Our data suggest that the 5′ external transcribed spacer is shortened by AtXRN2 prior to cleavage at site P and that this initial exonucleolytic trimming is required to expose site P for subsequent endonucleolytic processing by the U3 snoRNP complex. We also show that some rRNA precursors and excised spacer fragments that accumulate in the absence of AtXRN2 and AtXRN3 are polyadenylated, indicating that these nucleases contribute to polyadenylation-dependent nuclear RNA surveillance.

Lsm Proteins Are Required for Normal Processing of Pre-tRNAs and Their Efficient Association with La-Homologous Protein Lhp1p

ABSTRACT Depletion of any of the five essential proteins Lsm2p to Lsm5p and Lsm8p leads to strong accumulation of all tested unspliced pre-tRNA species, as well as accumulation of 5′ and 3′ unprocessed species. Aberrant 3′-extended pre-tRNAs were detected, presumably due to stabilization of transcripts that fail to undergo correct transcription termination, and the accumulation of truncated tRNA fragments was also observed. Tandem affinity purification-tagged Lsm3p was associated with pre-tRNA primary transcripts and, less efficiently, with other unspliced pre-tRNA intermediates but not mature tRNAs. Association of the Saccharomyces cerevisiae La homologue Lhp1p with pre-tRNAs was reduced approximately threefold on depletion of Lsm3p or Lsm5p. The association of Lhp1p with larger RNA polymerase III transcripts, pre-RNase P RNA and the signal recognition particle RNA (scR1), was more drastically reduced. The impaired pre-tRNA processing seen on Lsm depletion is not, however, due solely to reduced Lhp1p association as evidenced by analysis of lhp1-Δ strains depleted of Lsm3p or Lsm5p. These data are consistent with roles for an Lsm complex as a chaperone that facilitates the efficient association of pre-tRNA processing factors with their substrates.

Nuclear Pre-mRNA Decapping and 5 Degradation in Yeast Require the Lsm2-8p Complex

ABSTRACT Previous analyses have identified related cytoplasmic Lsm1-7p and nuclear Lsm2-8p complexes. Here we report that mature heat shock and MET mRNAs that are trapped in the nucleus due to a block in mRNA export were strongly stabilized in strains lacking Lsm6p or the nucleus-specific Lsm8p protein but not by the absence of the cytoplasmic Lsm1p. These nucleus-restricted mRNAs remain polyadenylated until their degradation, indicating that nuclear mRNA degradation does not involve the incremental deadenylation that is a key feature of cytoplasmic turnover. Lsm8p can be UV cross-linked to nuclear poly(A)+ RNA, indicating that an Lsm2-8p complex interacts directly with nucleus-restricted mRNA. Analysis of pre-mRNAs that contain intronic snoRNAs indicates that their 5′ degradation is specifically inhibited in strains lacking any of the Lsm2-8p proteins but Lsm1p. Nucleus-restricted mRNAs and pre-mRNA degradation intermediates that accumulate in lsm mutants remain 5′ capped. We conclude that the Lsm2-8p complex normally targets nuclear RNA substrates for decapping.

Apoptotic signals induce specific degradation of ribosomal RNA in yeast

Organisms exposed to reactive oxygen species, generated endogenously during respiration or by environmental conditions, undergo oxidative stress. Stress response can either repair the damage or activate one of the programmed cell death (PCD) mechanisms, for example apoptosis, and finally end in cell death. One striking characteristic, which accompanies apoptosis in both vertebrates and yeast, is a fragmentation of cellular DNA and mammalian apoptosis is often associated with degradation of different RNAs. We show that in yeast exposed to stimuli known to induce apoptosis, such as hydrogen peroxide, acetic acid, hyperosmotic stress and ageing, two large subunit ribosomal RNAs, 25S and 5.8S, became extensively degraded with accumulation of specific intermediates that differ slightly depending on cell death conditions. This process is most likely endonucleolytic, is correlated with stress response, and depends on the mitochondrial respiratory status: rRNA is less susceptible to degradation in respiring cells with functional defence against oxidative stress. In addition, RNA fragmentation is independent of two yeast apoptotic factors, metacaspase Yca1 and apoptosis-inducing factor Aif1, but it relies on the apoptotic chromatin condensation induced by histone H2B modifications. These data describe a novel phenotype for certain stress- and ageing-related PCD pathways in yeast.

The P15-loop of Escherichia coli RNase P RNA is an autonomous divalent metal ion binding domain.

We have studied the structure and divalent metal ion binding of a domain of the ribozyme RNase P RNA that is involved in base pairing with its substrate. Our data suggest that the folding of this internal loop, the P15-loop, is similar irrespective of whether it is part of the full-length ribozyme or part of a model RNA molecule. We also conclude that this element constitutes an autonomous divalent metal ion binding domain of RNase P RNA and our data suggest that certain specific chemical groups within the P15-loop participate in coordination of divalent metal ions. Substitutions of the Sp- and Rp-oxygens with sulfur at a specific position in this loop result in a 2.5-5-fold less active ribozyme, suggesting that Mg2+ binding at this position contributes to function. Our findings strengthen the concept that small RNA building blocks remain basically unchanged when removed from their structural context and thus can be used as models for studies of their potential function and structure within native RNA molecules.