Precursor-product discrimination by La protein during tRNA metabolism

Intramural Research Program, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, USA.
Nature Structural & Molecular Biology (Impact Factor: 13.31). 05/2009; 16(4):430-7. DOI: 10.1038/nsmb.1573
Source: PubMed


La proteins bind pre-tRNAs at their UUU-3'OH ends, facilitating their maturation. Although the mechanism by which La binds pre-tRNA 3' trailers is known, the function of the RNA binding beta-sheet surface of the RNA-recognition motif (RRM1) is unknown. How La dissociates from UUU-3'OH-containing trailers after 3' processing is also unknown. Here we show that La preferentially binds pre-tRNAs over processed tRNAs or 3' trailer products through coupled use of two sites: one on the La motif and another on the RRM1 beta-surface that binds elsewhere on tRNA. Two sites provide stable pre-tRNA binding, whereas the processed tRNA and 3' trailer are released from their single sites relatively fast. RRM1 loop-3 mutations decrease affinity for pre-tRNA and tRNA, but not for the UUU-3'OH trailer, and impair tRNA maturation in vivo. We propose that RRM1 functions in activities that are more complex than UUU-3'OH binding. Accordingly, the RRM1 mutations also impair an RNA chaperone activity of La. The results suggest how La distinguishes precursor from product RNAs, allowing it to recycle onto a new pre-tRNA.

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    • "The pre-tRNA chaperone La protein specifically binds to pre-tRNAs in a 3′ oligo(U) lengthdependent manner. Therefore, increased 3′ oligo(U) length leads to higher affinity binding of La protein to the pre-tRNAs and thereby protects them from 3′ exonucleolytic digestion and enhances their folding and maturation ((Huang et al., 2006, 2005; Bayfield and Maraia, 2009), reviewed in (Maraia and Lamichhane, 2011b)). Natural heterogeneity of 3′ oligo(U) length exists in pre-tRNAs in wild type S. pombe cells (Huang et al., 2005). "
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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.
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    • "We suggest naming this domain xRRM for atypical RRM with C-terminal extension and identifying the recognition motif on b2 as RNP3. Genuine La proteins bind the 3 0 UUU OH of newly synthesized Pol III and some Pol II noncoding RNA transcripts with the LAM and RRM1 domains, protecting them from degradation (Bayfield and Maraia, 2009; Kotik-Kogan et al., 2008; Teplova et al., 2006; Wolin and Cedervall, 2002). La proteins also function as RNA chaperones for correct folding of diverse RNAs such as some tRNAs, snRNAs, snoRNAs, and mRNA structural elements (Kucera et al., 2011; Wolin and Cedervall, 2002). "
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    • "This mode of RNA binding is unusual and intriguing since it uses neither of the canonical RNA-binding surfaces—the winged-helix of the LaM or the β-sheet surface of RRM1—which may therefore be potentially available to interact with other portions of larger RNA ligands. Indeed, recognition of 3′ oligoU sequences appears to be only one facet of the RNA interactions made by La and recent investigations of the functional interaction with pre-tRNA targets suggest that as well as clamping onto the 3′ oligoU trailer the protein establishes additional points of contact with the RNA (15–17). "
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