Lsm Proteins are required for normal processing and stability of ribosomal RNAs

Wellcome Trust Centre for Cell Biology, Swann Building, King's Buildings, University of Edinburgh, United Kingdom.
Journal of Biological Chemistry (Impact Factor: 4.57). 02/2003; 278(4):2147-56. DOI: 10.1074/jbc.M208856200
Source: PubMed

ABSTRACT 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.

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    • "A difference was however noted in that at the late time point of depletion of 22 h, the DKH core and G207A mutations (Fig. 2A, lanes 9,18, respectively ) accumulated a novel RNA that extended from site D to B 2 (data not shown). The D-B 2 RNA was previously reported in strains genetically depleted for Sm-like proteins and is indicative of alterations in pre-rRNA processing kinetics (Kufel et al. 2003). "
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    ABSTRACT: Eukaryotic ribosome synthesis is a highly dynamic process that involves the transient association of scores of trans-acting factors to nascent pre-ribosomes. Many ribosome synthesis factors are nucleocytoplasmic shuttling proteins that engage the assembly pathway at early nucleolar stages and escort pre-ribosomes to the nucleoplasm and/or the cytoplasm. Here, we report that two 40S ribosome synthesis factors, the KH-domain protein DIM2 and the HEAT-repeats/Armadillo-domain and export factor RRP12, are nucleolar restricted upon nutritional, osmotic, and oxidative stress. Nucleolar entrapment of DIM2 and RRP12 was triggered by rapamycin treatment and was under the strict control of the target of rapamycin (TOR) signaling cascade. DIM2 binds pre-rRNAs directly through its KH domain at the 5'-end of ITS1 (D-A(2) segment) and, consistent with its requirements in early nucleolar pre-rRNA processing, is required for efficient cotranscriptional ribosome assembly. The substitution of a single and highly conserved amino acid (G207A) within the KH motif is sufficient to inhibit pre-rRNA processing in a fashion similar to genetic depletion of DIM2. DIM2 carries an evolutionarily conserved putative nuclear export sequence (NES) at its carboxyl-terminal end that is required for efficient pre-40S ribosome export. Strikingly, DIM2 and RRP12 are both involved in the nucleocytoplasmic translocation of pre-ribosomes, suggesting that this step in the ribosome assembly pathway has been selected as a regulatory target for the TOR pathway.
    RNA 09/2008; 14(10):2061-73. DOI:10.1261/rna.1176708 · 4.62 Impact Factor
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    • "They may therefore also have indirect effects on tRNA modification. Lsm6p has in fact also been implicated in tRNA processing (Kufel et al. 2003). "
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    ABSTRACT: We have screened a collection of haploid yeast knockout strains for increased sensitivity to 5-fluorouracil (5-FU). A total of 138 5-FU sensitive strains were found. Mutants affecting rRNA and tRNA maturation were particularly sensitive to 5-FU, with the tRNA methylation mutant trm10 being the most sensitive mutant. This is intriguing since trm10, like many other tRNA modification mutants, lacks a phenotype under normal conditions. However, double mutants for nonessential tRNA modification enzymes are frequently temperature sensitive, due to destabilization of hypomodified tRNAs. We therefore tested if the sensitivity of our mutants to 5-FU is affected by the temperature. We found that the cytotoxic effect of 5-FU is strongly enhanced at 38 degrees C for tRNA modification mutants. Furthermore, tRNA modification mutants show similar synthetic interactions for temperature sensitivity and sensitivity to 5-FU. A model is proposed for how 5-FU kills these mutants by reducing the number of tRNA modifications, thus destabilizing tRNA. Finally, we found that also wild-type cells are temperature sensitive at higher concentrations of 5-FU. This suggests that tRNA destabilization contributes to 5-FU cytotoxicity in wild-type cells and provides a possible explanation why hyperthermia can enhance the effect of 5-FU in cancer therapy.
    RNA 05/2008; 14(4):666-74. DOI:10.1261/rna.966208 · 4.62 Impact Factor
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    • "The second conserved sequence, which spans a region between aa 1000 and 1090, shows a high similarity with the active domain of SceSnp3p (Fig. 4B). This protein has an important function in binding and stabilization of the 3 end of the spliceosomal U6 snRNA (Kufel et al., 2003). AniPso2p FA shows a highly conserved Pso2p A domain situated around aa 1440 and 1820 (third domain, Fig. 4B). "
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