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.
    Gene 11/2014; 556(1). DOI:10.1016/j.gene.2014.11.034 · 2.14 Impact Factor
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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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    ABSTRACT: Telomerase is a ribonucleoprotein complex essential for maintenance of telomere DNA at linear chromosome ends. The catalytic core of Tetrahymena telomerase comprises a ternary complex of telomerase RNA (TER), telomerase reverse transcriptase (TERT), and the essential La family protein p65. NMR and crystal structures of p65 C-terminal domain and its complex with stem IV of TER reveal that RNA recognition is achieved by a combination of single- and double-stranded RNA binding, which induces a 105° bend in TER. The domain is a cryptic, atypical RNA recognition motif with a disordered C-terminal extension that forms an α helix in the complex necessary for hierarchical assembly of TERT with p65-TER. This work provides the first structural insight into biogenesis and assembly of TER with a telomerase-specific protein. Additionally, our studies define a structurally homologous domain (xRRM) in genuine La and LARP7 proteins and suggest a general mode of RNA binding for biogenesis of their diverse RNA targets.
    Molecular cell 06/2012; 47(1):16-26. DOI:10.1016/j.molcel.2012.05.018 · 14.02 Impact Factor
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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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    ABSTRACT: Human La protein is an essential factor in the biology of both coding and non-coding RNAs. In the nucleus, La binds primarily to 3' oligoU containing RNAs, while in the cytoplasm La interacts with an array of different mRNAs lacking a 3' UUU(OH) trailer. An example of the latter is the binding of La to the IRES domain IV of the hepatitis C virus (HCV) RNA, which is associated with viral translation stimulation. By systematic biophysical investigations, we have found that La binds to domain IV using an RNA recognition that is quite distinct from its mode of binding to RNAs with a 3' UUU(OH) trailer: although the La motif and first RNA recognition motif (RRM1) are sufficient for high-affinity binding to 3' oligoU, recognition of HCV domain IV requires the La motif and RRM1 to work in concert with the atypical RRM2 which has not previously been shown to have a significant role in RNA binding. This new mode of binding does not appear sequence specific, but recognizes structural features of the RNA, in particular a double-stranded stem flanked by single-stranded extensions. These findings pave the way for a better understanding of the role of La in viral translation initiation.
    Nucleic Acids Research 02/2012; 40(3):1381-94. DOI:10.1093/nar/gkr890 · 9.11 Impact Factor
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