Conservation of a Masked Nuclear Export Activity of La Proteins and Its Effects on tRNA Maturation

Intramural Research Program, National Institute of Child Health and Human Development, U.S. National Institutes of Health, Bethesda, MD 20892-2426, USA.
Molecular and Cellular Biology (Impact Factor: 4.78). 06/2007; 27(9):3303-12. DOI: 10.1128/MCB.00026-07
Source: PubMed


La is an RNA-processing-associated phosphoprotein so highly conserved that the human La protein (hLa) can replace the tRNA-processing function of the fission yeast La protein (Sla1p) in vivo. La proteins contain multiple trafficking elements that support interactions with RNAs in different subcellular locations. Prior data indicate that deletion of a nuclear retention element (NRE) causes nuclear export of La and dysfunctional processing of associated pre-tRNAs that are spliced but 5' and 3' unprocessed, with an accompanying decrease in tRNA-mediated suppression, in fission yeast. To further pursue these observations, we first identified conserved residues in the NREs of hLa and Sla1p that when substituted mimic the NRE deletion phenotype. NRE-defective La proteins then deleted of other motifs indicated that RNA recognition motif 1 (RRM1) is required for nuclear export. Mutations of conserved RRM1 residues restored nuclear accumulation of NRE-defective La proteins. Some RRM1 mutations restored nuclear accumulation, prevented disordered pre-tRNA processing, and restored suppression, indicating that the tRNA-related activity of RRM1 and its nuclear export activity could be functionally separated. When mapped onto an hLa structure, the export-sensitive residues comprised surfaces distinct from the RNA-binding surface of RRM1. The data indicate that the NRE has been conserved to mask or functionally override an equally conserved nuclear export activity of RRM1. The data suggest that conserved elements mediate nuclear retention, nuclear export, and RNA-binding activities of the multifunctional La protein and that their interrelationship contributes to the ability of La to engage its different classes of RNA ligands in different cellular locations.

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Available from: Robert Intine, Apr 28, 2014
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    • "For these tRNAs, consistent with biochemical findings that indicate that La binding inhibits 3′ processing by RNase Z (Nashimoto et al., 2001), the pre-tRNA 3′ end determines processing order in the presence of La and when La is absent, 3′ end processing occurs first (Yoo and Wolin, 1997). That the 3′ end is a major determinant of transcript fate is supported by the compelling finding that La proteins bearing mutations that activate its premature nuclear export carry their pre-tRNA ligands with their intact 5′ leaders and 3′ trailers, to the cytoplasm, bypassing processing by RNases P and Z (Bayfield et al., 2007; Intine et al., 2002). "
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    ABSTRACT: Suppressor tRNAs bear anticodon mutations that allow them to decode premature stop codons in metabolic marker gene mRNAs, that can be used as in vivo reporters of functional tRNA biogenesis. Here, we review key components of a suppressor tRNA system specific to Schizosaccharomyces pombe and its adaptations for use to study specific steps in tRNA biogenesis. Eukaryotic tRNA biogenesis begins with transcription initiation by RNA polymerase (pol) III. The nascent pre-tRNAs must undergo folding, 5' and 3' processing to remove the leader and trailer, nuclear export, and splicing if applicable, while multiple complex chemical modifications occur throughout the process. We review evidence that precursor-tRNA processing begins with transcription termination at the oligo(T) terminator element, which forms a 3' oligo(U) tract on the nascent RNA, a sequence-specific binding site for the RNA chaperone, La protein. The processing pathway bifurcates depending on a poorly understood property of pol III termination that determines the 3' oligo(U) length and therefore the affinity for La. We thus review the pol III termination process and the factors involved including advances using gene-specific random mutagenesis by dNTP analogs that identify key residues important for transcription termination in certain pol III subunits. The review ends with a 'technical approaches' section that includes a parts lists of suppressor-tRNA alleles, strains and plasmids, and graphic examples of its diverse uses. Published by Elsevier B.V.
    Full-text · Article · Nov 2014 · Gene
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    • "Its principal RNA-binding activity, UUU-3′-OH binding, is mediated primarily by the LAM to protect against 3′ exonucleases and promote 3′ end processing and RNP maturation. Mutations in human La (hLa) or Schizosaccharomyces pombe La, Sla1p, that disrupt RNA 3′ end binding or intracellular trafficking cause tRNA processing defects consistent with dysfunctional molecular chaperone activity (42,43). "
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    ABSTRACT: We used a genetic screen based on tRNA-mediated suppression (TMS) in a Schizosaccharomyces pombe La protein (Sla1p) mutant. Suppressor pre-tRNASerUCA-C47:6U with a debilitating substitution in its variable arm fails to produce tRNA in a sla1-rrm mutant deficient for RNA chaperone-like activity. The parent strain and spontaneous mutant were analyzed using Solexa sequencing. One synonymous single-nucleotide polymorphism (SNP), unrelated to the phenotype, was identified. Further sequence analyses found a duplication of the tRNASerUCA-C47:6U gene, which was shown to cause the phenotype. Ninety percent of 28 isolated mutants contain duplicated tRNASerUCA-C47:6U genes. The tRNA gene duplication led to a disproportionately large increase in tRNASerUCA-C47:6U levels in sla1-rrm but not sla1-null cells, consistent with non-specific low-affinity interactions contributing to the RNA chaperone-like activity of La, similar to other RNA chaperones. Our analysis also identified 24 SNPs between ours and S. pombe 972h- strain yFS101 that was recently sequenced using Solexa. By including mitochondrial (mt) DNA in our analysis, overall coverage increased from 52% to 96%. mtDNA from our strain and yFS101 shared 14 mtSNPs relative to a ‘reference’ mtDNA, providing the first identification of these S. pombe mtDNA discrepancies. Thus, strain-specific and spontaneous phenotypic mutations can be mapped in S. pombe by Solexa sequencing.
    Full-text · Article · Feb 2011 · Nucleic Acids Research
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    • "The La-related proteins (LARPs) comprise an evolutionarily conserved group of factors with presumed roles in RNA metabolism (Bayfield et al. 2007; Bousquet-Antonelli and Deragon 2009). Thus far, seven different LARPs have been identified in humans (termed LARP1, 1B, 3, 4A, 4B, 6, and 7) based on their homology with the genuine La protein (also termed LARP3 according to the new nomenclature), the founding member of this family (Wolin and Cedervall 2002). "
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    ABSTRACT: La-related proteins (LARPs) belong to an evolutionarily conserved family of factors with predicted roles in RNA metabolism. Here, we have analyzed the cellular interactions and function of LARP4B, a thus far uncharacterized member of the LARP family. We show that LARP4B is a cytosolic protein that accumulates upon arsenite treatment in cellular stress granules. Biochemical experiments further uncovered an interaction of LARP4B with the cytosolic poly(A) binding protein 1 (PABPC1) and the receptor for activated C Kinase (RACK1), a component of the 40S ribosomal subunit. Under physiological conditions, LARP4B co-sedimented with polysomes in cellular extracts, suggesting a role in translation. In agreement with this notion, overexpression of LARP4B stimulated protein synthesis, whereas knockdown of the factor by RNA interference impaired translation of a large number of cellular mRNAs. In sum, we identified LARP4B as a stimulatory factor of translation. We speculate that LARP4B exerts its function by bridging mRNA factors of the 3' end with initiating ribosomes.
    Full-text · Article · Aug 2010 · RNA
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