tRNAs marked with CCACCA are targeted for degradation

Koch Institute for Integrative Cancer Research and Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Science (Impact Factor: 33.61). 11/2011; 334(6057):817-21. DOI: 10.1126/science.1213671
Source: PubMed


The CCA-adding enzyme [ATP(CTP):tRNA nucleotidyltransferase] adds CCA to the 3' ends of transfer RNAs (tRNAs), a critical step in tRNA biogenesis that generates the amino acid attachment site. We found that the CCA-adding enzyme plays a key role in tRNA quality control by selectively marking structurally unstable tRNAs and tRNA-like small RNAs for degradation. Instead of adding CCA to the 3' ends of these transcripts, CCA-adding enzymes from all three kingdoms of life add CCACCA. In addition, hypomodified mature tRNAs are subjected to CCACCA addition as part of a rapid tRNA decay pathway in vivo. We conjecture that CCACCA addition is a universal mechanism for controlling tRNA levels and preventing errors in translation.

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Available from: Joseph Whipple, Mar 26, 2014
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    • "The CCA-3 is also required for tRNA quality control. The tandem C 74 C 75 A 76 C 77 C 78 A 79 -3 sequence, added onto the 3 -end of tRNA, acts as a degradation signal for dysfunctional tRNA molecules (Wilusz et al., 2011). The CCA-3 is synthesized and/or repaired by the CCA-adding enzyme, CTP:(ATP) tRNA nucleotidyltransferase (NT), using CTP and ATP as substrates (Deutscher, 1990; Weiner, 2004). "
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    ABSTRACT: The universal 3'-terminal CCA sequence of tRNA is built and/or synthesized by the CCA-adding enzyme, CTP:(ATP) tRNA nucleotidyltransferase. This RNA polymerase has no nucleic acid template, but faithfully synthesizes the defined CCA sequence on the 3'-terminus of tRNA at one time, using CTP and ATP as substrates. The mystery of CCA-addition without a nucleic acid template by unique RNA polymerases has long fascinated researchers in the field of RNA enzymology. In this review, the mechanisms of RNA polymerization by the remarkable CCA-adding enzyme and its related enzymes are presented, based on their structural features.
    Frontiers in Genetics 02/2014; 5:36. DOI:10.3389/fgene.2014.00036
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    • "The invariant CCA-3 0 is required for amino acid attachment onto the 3 0 end of the tRNA by aminoacyl-tRNA synthases (Sprinzl and Cramer, 1979), and for peptide-bond formation on ribosomes (Green and Noller, 1997; Kim and Green, 1999; Nissen et al., 2000). The CCA-3 0 is also required for tRNA quality control (Wilusz et al., 2011). "
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    ABSTRACT: The 3'-terminal CCA (CCA-3' at positions 74-76) of tRNA is synthesized by CCA-adding enzyme using CTP and ATP as substrates, without a nucleic acid template. In Aquifex aeolicus, CC-adding and A-adding enzymes collaboratively synthesize the CCA-3'. The mechanism of CCA-3' synthesis by these two enzymes remained obscure. We now present crystal structures representing CC addition onto tRNA by A. aeolicus CC-adding enzyme. After C74 addition in an enclosed active pocket and pyrophosphate release, the tRNA translocates and rotates relative to the enzyme, and C75 addition occurs in the same active pocket as C74 addition. At both the C74-adding and C75-adding stages, CTP is selected by Watson-Crick-like hydrogen bonds between the cytosine of CTP and conserved Asp and Arg residues in the pocket. After C74C75 addition and pyrophosphate release, the tRNA translocates further and drops off the enzyme, and the CC-adding enzyme terminates RNA polymerization.
    Structure 12/2013; 22(2). DOI:10.1016/j.str.2013.12.002 · 5.62 Impact Factor
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    • "To comprehensively define the 3′-hydroxyl ends of endogenous RNA molecules in HEK293T cells, we adapted an RNA ligase-mediated 3′ RACE strategy coupled to deep sequencing (Figure 1). While similar methods have been previously reported [33], our protocol incorporated a few modifications. Precise 3′ terminal nucleotides were demarcated by ligation of whole cell RNA preparations with four distinct oligoribonucleotide appendices, each containing a different 5′ terminal base and internal barcode to minimize structural bias during this reaction [32,35]. "
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    ABSTRACT: Post-transcriptional 3[prime] end processing is a key component of RNA regulation. The abundant and essential RNA subunit of RNase MRP has been proposed to function in three distinct cellular compartments and therefore may utilize this mode of regulation. Here we employ 3[prime] RACE coupled with high-throughput sequencing to characterize the 3[prime] terminal sequences of human MRP RNA and other noncoding RNAs that form RNP complexes. The 3[prime] terminal sequence of MRP RNA from HEK293T cells has a distinctive distribution of genomically encoded termini (including an assortment of U residues) with a portion of these selectively tagged by oligo(A) tails. This profile contrasts with the relatively homogenous 3[prime] terminus of an in vitro transcribed MRP RNA control and the differing 3[prime] terminal profiles of U3 snoRNA, RNase P RNA, and telomerase RNA (hTR). 3[prime] RACE coupled with deep sequencing provides a valuable framework for the functional characterization of 3[prime] terminal sequences of noncoding RNAs.
    BMC Molecular Biology 09/2013; 14(1):23. DOI:10.1186/1471-2199-14-23 · 2.19 Impact Factor
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