Use of Specific Chemical Reagents for Detection of Modified Nucleotides in RNA

Naturally occurring cellular RNAs contain an impressive number of chemically distinct modified residues which appear posttranscriptionally, as a result of specific action of the corresponding RNA modification enzymes. Over 100 different chemical modifications have been identified and characterized up to now. Identification of the chemical nature and exact position of these modifications is typically based on 2D-TLC analysis of nucleotide digests, on HPLC coupled with mass spectrometry, or on the use of primer extension by reverse transcriptase. However, many modified nucleotides are silent in reverse transcription, since the presence of additional chemical groups frequently does not change base-pairing properties. In this paper, we give a summary of various chemical approaches exploiting the specific reactivity of modified nucleotides in RNA for their detection

Long 3′-UTRs target wild-type mRNAs for nonsense-mediated mRNA decay in Saccharomyces cerevisiae

The nonsense-mediated mRNA decay (NMD) pathway, present in most eukaryotic cells, is a specialized pathway that leads to the recognition and rapid degradation of mRNAs with premature termination codons and, importantly, some wild-type mRNAs. Earlier studies demonstrated that aberrant mRNAs with artificially extended 3′-untranslated regions (3′-UTRs) are degraded by NMD. However, the extent to which wild-type mRNAs with long 3′-UTRs are degraded by NMD is not known. We used a global approach to identify wild-type mRNAs in Saccharomyces cerevisiae that have longer than expected 3′-UTRs, and of these mRNAs tested, 91% were degraded by NMD. We demonstrate for the first time that replacement of the natural, long 3′-UTR from wild-type PGA1 mRNA, which encodes a protein that is important for cell wall biosynthesis, with a short 3′-UTR renders it immune to NMD. The natural PGA1 3′-UTR is sufficient to target a NMD insensitive mRNA for decay by the NMD pathway. Finally, we show that nmd mutants are sensitive to Calcofluor White, which suggests that the regulation of PGA1 and other cell wall biosynthesis proteins by NMD is physiologically significant

The RRM domain in GW182 proteins contributes to miRNA-mediated gene silencing

Proteins of the GW182 family interact with Argonaute proteins and are required for miRNA-mediated gene silencing. These proteins contain two structural domains, an ubiquitin-associated (UBA) domain and an RNA recognition motif (RRM), embedded in regions predicted to be unstructured. The structure of the RRM of Drosophila melanogaster GW182 reveals that this domain adopts an RRM fold, with an additional C-terminal α-helix. The helix lies on the β-sheet surface, generally used by these domains to bind RNA. This, together with the absence of aromatic residues in the conserved RNP1 and RNP2 motifs, and the lack of general affinity for RNA, suggests that the GW182 RRM does not bind RNA. The domain may rather engage in protein interactions through an unusual hydrophobic cleft exposed on the opposite face of the β-sheet. We further show that the GW182 RRM is dispensable for P-body localization and for interaction of GW182 with Argonaute-1 and miRNAs. Nevertheless, its deletion impairs the silencing activity of GW182 in a miRNA target-specific manner, indicating that this domain contributes to silencing. The conservation of structural and surface residues suggests that the RRM domain adopts a similar fold with a related function in insect and vertebrate GW182 family members

Posttranscriptional Gene Regulation by Spatial Rearrangement of the 3′ Untranslated Region

Translation termination at premature termination codons (PTCs) triggers degradation of the aberrant mRNA, but the mechanism by which a termination event is defined as premature is still unclear. Here we show that the physical distance between the termination codon and the poly(A)-binding protein PABPC1 is a crucial determinant for PTC recognition in human cells. “Normal” termination codons can trigger nonsense-mediated mRNA decay (NMD) when this distance is extended; and vice versa, NMD can be suppressed by folding the poly(A) tail into proximity of a PTC or by tethering of PABPC1 nearby a PTC, indicating an evolutionarily conserved function of PABPC1 in promoting correct translation termination and antagonizing activation of NMD. Most importantly, our results demonstrate that spatial rearrangements of the 3′ untranslated region can modulate the NMD pathway and thereby provide a novel mechanism for posttranscriptional gene regulation

Dhx34 and Nbas function in the NMD pathway and are required for embryonic development in zebrafish

The nonsense-mediated mRNA decay (NMD) pathway is a highly conserved surveillance mechanism that is present in all eukaryotes. It prevents the synthesis of truncated proteins by selectively degrading mRNAs harbouring premature termination codons (PTCs). The core NMD effectors were originally identified in genetic screens in Saccharomyces cerevisae and in the nematode Caenorhabditis elegans, and subsequently by homology searches in other metazoans. A genome-wide RNAi screen in C. elegans resulted in the identification of two novel NMD genes that are essential for proper embryonic development. Their human orthologues, DHX34 and NAG/NBAS, are required for NMD in human cells. Here, we find that the zebrafish genome encodes orthologues of DHX34 and NAG/NBAS. We show that the morpholino-induced depletion of zebrafish Dhx34 and Nbas proteins results in severe developmental defects and reduced embryonic viability. We also found that Dhx34 and Nbas are required for degradation of PTC-containing mRNAs in zebrafish embryos. The phenotypes observed in both Dhx34 and Nbas morphants are similar to defects in Upf1, Smg-5- or Smg-6- depleted embryos, suggesting that these factors affect the same pathway and confirming that zebrafish embryogenesis requires an active NMD pathway

Combined in silico and experimental identification of the Pyrococcus abyssi H/ACA sRNAs and their target sites in ribosomal RNAs

How far do H/ACA sRNPs contribute to rRNA pseudouridylation in Archaea was still an open question. Hence here, by computational search in three Pyrococcus genomes, we identified seven H/ACA sRNAs and predicted their target sites in rRNAs. In parallel, we experimentally identified 17 Ψ residues in P. abyssi rRNAs. By in vitro reconstitution of H/ACA sRNPs, we assigned 15 out of the 17 Ψ residues to the 7 identified H/ACA sRNAs: one H/ACA motif can guide up to three distinct pseudouridylations. Interestingly, by using a 23S rRNA fragment as the substrate, one of the two remaining Ψ residues could be formed in vitro by the aCBF5/aNOP10/aGAR1 complex without guide sRNA. Our results shed light on structural constraints in archaeal H/ACA sRNPs: the length of helix H2 is of 5 or 6 bps, the distance between the ANA motif and the targeted U residue is of 14 or 15 nts, and the stability of the interaction formed by the substrate rRNA and the 3′-guide sequence is more important than that formed with the 5′-guide sequence. Surprisingly, we showed that a sRNA–rRNA interaction with the targeted uridine in a single-stranded 5′-UNN-3′ trinucleotide instead of the canonical 5′-UN-3′ dinucleotide is functional

Identification of human tRNA:m(5)C methyltransferase catalysing intron-dependent m(5)C formation in the first position of the anticodon of the [Formula: see text]

We identified a human orthologue of tRNA:m(5)C methyltransferase from Saccharomyces cerevisiae, which has been previously shown to catalyse the specific modification of C(34) in the intron-containing yeast [Formula: see text]. Using transcripts of intron-less and intron-containing human [Formula: see text] genes as substrates, we have shown that m(5)C(34) is introduced only in the intron-containing tRNA precursors when the substrates were incubated in the HeLa extract. m(5)C(34) formation depends on the nucleotide sequence surrounding the wobble cytidine and on the structure of the prolongated anticodon stem. Expression of the human Trm4 (hTrm4) cDNA in yeast partially complements the lack of the endogenous Trm4p enzyme. The yeast extract prepared from the strain deprived of the endogenous TRM4 gene and transformed with hTrm4 cDNA exhibits the same activity and substrate specificity toward human pre-tRNA(Leu) transcripts as the HeLa extract. The hTrm4 MTase has a much narrower specificity against the yeast substrates than its yeast orthologue: human enzyme is not able to form m(5)C at positions 48 and 49 of human and yeast tRNA precursors. To our knowledge, this is the first report showing intron-dependent methylation of human [Formula: see text] and identification of human gene encoding tRNA methylase responsible for this reaction

The GW/WG repeats of Drosophila GW182 function as effector motifs for miRNA-mediated repression

The control of messenger RNA (mRNA) function by micro RNAs (miRNAs) in animal cells requires the GW182 protein. GW182 is recruited to the miRNA repression complex via interaction with Argonaute protein, and functions downstream to repress protein synthesis. Interaction with Argonaute is mediated by GW/WG repeats, which are conserved in many Argonaute-binding proteins involved in RNA interference and miRNA silencing, from fission yeast to mammals. GW182 contains at least three effector domains that function to repress target mRNA. Here, we analyze the functions of the N-terminal GW182 domain in repression and Argonaute1 binding, using tethering and immunoprecipitation assays in Drosophila cultured cells. We demonstrate that its function in repression requires intact GW/WG repeats, but does not involve interaction with the Argonaute1 protein, and is independent of the mRNA polyadenylation status. These results demonstrate a novel role for the GW/WG repeats as effector motifs in miRNA-mediated repression

HPat provides a link between deadenylation and decapping in metazoa

A proline-rich region in the Drosophila Pat1 homologue works with the protein's C-terminal domain to recruit decapping and deadenylase complexes to target mRNAs

Ex vivo correction of selenoprotein N deficiency in rigid spine muscular dystrophy caused by a mutation in the selenocysteine codon

Premature termination of translation due to nonsense mutations is a frequent cause of inherited diseases. Therefore, many efforts were invested in the development of strategies or compounds to selectively suppress this default. Selenoproteins are interesting candidates considering the idiosyncrasy of the amino acid selenocysteine (Sec) insertion mechanism. Here, we focused our studies on SEPN1, a selenoprotein gene whose mutations entail genetic disorders resulting in different forms of muscular diseases. Selective correction of a nonsense mutation at the Sec codon (UGA to UAA) was undertaken with a corrector tRNASec that was engineered to harbor a compensatory mutation in the anticodon. We demonstrated that its expression restored synthesis of a full-length selenoprotein N both in HeLa cells and in skin fibroblasts from a patient carrying the mutated Sec codon. Readthrough of the UAA codon was effectively dependent on the Sec insertion machinery, therefore being highly selective for this gene and unlikely to generate off-target effects. In addition, we observed that expression of the corrector tRNASec stabilized the mutated SEPN1 transcript that was otherwise more subject to degradation. In conclusion, our data provide interesting evidence that premature termination of translation due to nonsense mutations is amenable to correction, in the context of the specialized selenoprotein synthesis mechanism