Translon: a single term for translated regions.
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Letter↗
Biomedical subjects
Joanna Kufel
Publications and source records attributed to Joanna Kufel.
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 degrees 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.
Blotting, Northern↗
Substrate discrimination in RNase P RNA-mediated cleavage: importance of the structural environment of the RNase P cleavage site.
Like the translational elongation factor EF-Tu, RNase P interacts with a large number of substrates where RNase P with its RNA subunit generates tRNAs with matured 5' termini by cleaving tRNA precursors immediately 5' of the residue at +1, i.e. at the position that corresponds to the first residue in tRNA. Most tRNAs carry a G+1C+72 base pair at the end of the aminoacyl acceptor-stem whereas in tRNA(Gln) G+1C+72 is replaced with U+1A+72. Here, we investigated RNase P RNA-mediated cleavage as a function of having G+1C+72 versus U+1A+72 in various substrate backgrounds, two full-size tRNA precursors (pre-tRNA(Gln) and pre-tRNA(Tyr)Su3) and a model RNA hairpin substrate (pATSer). Our data showed that replacement of G+1C+72 with U+1A+72 influenced ground state binding, cleavage efficiency under multiple and single turnover conditions in a substrate-dependent manner. Interestingly, we observed differences both in ground state binding and rate of cleavage comparing two full-size tRNA precursors, pre-tRNA(Gln) and pre-tRNA(Tyr)Su3. These findings provide evidence for substrate discrimination in RNase P RNA-mediated cleavage both at the level of binding, as previously observed for EF-Tu, as well as at the catalytic step. In our experiments where we used model substrate derivatives further indicated the importance of the +1/+72 base pair in substrate discrimination by RNase P RNA. Finally, we provide evidence that the structural architecture influences Mg2+ binding, most likely in its vicinity.
Base Pairing↗
Nuclear pre-mRNA decapping and 5' degradation in yeast require the Lsm2-8p complex.
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.
Cell Nucleus↗
A complex pathway for 3' processing of the yeast U3 snoRNA.
Mature U3 snoRNA in yeast is generated from the 3'-extended precursors by endonucleolytic cleavage followed by exonucleolytic trimming. These precursors terminate in poly(U) tracts and are normally stabilised by binding of the yeast La homologue, Lhp1p. We report that normal 3' processing of U3 requires the nuclear Lsm proteins. On depletion of any of the five essential proteins, Lsm2-5p or Lsm8p, the normal 3'-extended precursors to the U3 snoRNA were lost. Truncated fragments of both mature and pre-U3 accumulated in the Lsm-depleted strains, consistent with substantial RNA degradation. Pre-U3 species were co-precipitated with TAP-tagged Lsm3p, but the association with spliced pre-U3 was lost in strains lacking Lhp1p. The association of Lhp1p with pre-U3 was also reduced on depletion of Lsm3p or Lsm5p, indicating that binding of Lhp1p and the Lsm proteins is interdependent. In contrast, a tagged Sm-protein detectably co-precipitated spliced pre-U3 species only in strains lacking Lhp1p. We propose that the Lsm2-8p complex functions as a chaperone in conjunction with Lhp1p to stabilise pre-U3 RNA species during 3' processing. The Sm complex may function as a back-up to stabilise 3' ends that are not protected by Lhp1p.
Mutation↗
3'-processing of yeast tRNATrp precedes 5'-processing.
Previous analyses of eukaryotic pre-tRNAs processing have reported that 5'-cleavage by RNase P precedes 3'-maturation. Here we report that in contrast to all other yeast tRNAs analyzed to date, tRNA(Trp) undergoes 3'-maturation prior to 5'-cleavage. Despite its unusual processing pathway, pre-tRNA(Trp) resembles other pre-tRNAs, showing dependence on the essential Lsm proteins for normal processing and efficient association with the yeast La homolog, Lhp1p. tRNA(Trp) is also unusual in not requiring Lhp1p for 3' processing and stability. However, other Lhp1p-independent tRNAs, tRNA(2)(Lys) and tRNA(1)(Ile), follow the normal pathway of 5'-processing prior to 3-processing.
N-Terminal Acetyltransferase C↗
Lsm Proteins are required for normal processing and stability of ribosomal RNAs.
Depletion of any of the essential Lsm proteins, Lsm2-5p or Lsm8p, delayed pre-rRNA processing and led to the accumulation of many aberrant processing intermediates, indicating that an Lsm complex is required to maintain the normally strict order of processing events. In addition, high levels of degradation products derived from both precursors and mature rRNAs accumulated in Lsm-depleted strains. Depletion of the essential Lsm proteins reduced the apparent processivity of both 5' and 3' exonuclease activities involved in 5.8S rRNA processing, and the degradation intermediates that accumulated were consistent with inefficient 5' and 3' degradation. Many, but not all, pre-rRNA species could be coprecipitated with tagged Lsm3p, but not with tagged Lsm1p or non-tagged control strains, suggesting their direct interaction with an Lsm2-8p complex. We propose that Lsm proteins facilitate RNA protein interactions and structural changes required during ribosomal subunit assembly.
Base Sequence↗
Processing of 3'-extended read-through transcripts by the exosome can generate functional mRNAs.
Strains carrying rna14.1 and rna15.2 mutations are defective in pre-mRNA 3' cleavage, polyadenylation, and transcription termination. Long extended read-through transcripts generated in rna14.1 and rna15.2 strains are greatly stabilized by depletion of Rrp41p, a core component of the exosome complex or the RNA helicase Dob1p/Mtr4p. The absence of the nuclear-specific exosome component, Rrp6p, from the rna14.1 strain gave a very different phenotype. Short polyadenylated pre-mRNAs were strongly stabilized, and these were functional for translation. Production of these mRNAs was suppressed by depletion of Rrp41p, indicating that they are the products of exosome processing followed by uncoupled polyadenylation. The balance between complete degradation of 3'-unprocessed pre-mRNAs and their processing to functional mRNAs is regulated, with degradation favored on glucose media.
3' Untranslated Regions↗
Lsm proteins are required for normal processing of pre-tRNAs and their efficient association with La-homologous protein Lhp1p.
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-Delta 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.
Fungal Proteins↗