Biochemical characterization of mRNA capping enzyme from Faustovirus.

The mammalian mRNA 5' cap structures play important roles in cellular processes such as nuclear export, efficient translation, and evading cellular innate immune surveillance and regulating 5'-mediated mRNA turnover. Hence, installation of the proper 5' cap is crucial in therapeutic applications of synthetic mRNA. The core 5' cap structure, Cap-0, is generated by three sequential enzymatic activities: RNA 5' triphosphatase, RNA guanylyltransferase, and cap N7-guanine methyltransferase.

RNase H-based analysis of synthetic mRNA 5' cap incorporation.

Advances in mRNA synthesis and lipid nanoparticles technologies have helped make mRNA therapeutics and vaccines a reality. The 5' cap structure is a crucial modification required to functionalize synthetic mRNA for efficient protein translation in vivo and evasion of cellular innate immune responses. The extent of 5' cap incorporation is one of the critical quality attributes in mRNA manufacturing.

The yeast scavenger decapping enzyme DcpS and its application for in vitro RNA recapping.

Eukaryotic mRNAs are modified at their 5' end early during transcription by the addition of N7-methylguanosine (mG), which forms the "cap" on the first 5' nucleotide. Identification of the 5' nucleotide on mRNA is necessary for determination of the Transcription Start Site (TSS). We explored the effect of various reaction conditions on the activity of the yeast scavenger mRNA decapping enzyme DcpS and examined decapping of 30 chemically distinct cap structures varying the state of methylation, sugar, phosphate linkage, and base composition on 25mer RNA oligonucleotides.

Genome-wide profiling of the C. elegans dsRNAome.

Recent studies hint that endogenous dsRNA plays an unexpected role in cellular signaling. However, a complete understanding of endogenous dsRNA signaling is hindered by an incomplete annotation of dsRNA-producing genes. To identify dsRNAs expressed in Caenorhabditis elegans, we developed a bioinformatics pipeline that identifies dsRNA by detecting clustered RNA editing sites, which are strictly limited to long dsRNA substrates of Adenosine Deaminases that act on RNA (ADAR).

The yeast rapid tRNA decay pathway competes with elongation factor 1A for substrate tRNAs and acts on tRNAs lacking one or more of several modifications.

The structural and functional integrity of tRNA is crucial for translation. In the yeast Saccharomyces cerevisiae, certain aberrant pre-tRNA species are subject to nuclear surveillance, leading to 3' exonucleolytic degradation, and certain mature tRNA species are subject to rapid tRNA decay (RTD) if they are appropriately hypomodified or bear specific destabilizing mutations, leading to 5'-3' exonucleolytic degradation by Rat1 and Xrn1. Thus, trm8-Δ trm4-Δ strains are temperature sensitive due to lack of m(7)G(46) and m(5)C and the consequent RTD of tRNA(Val(AAC)), and tan1-Δ trm44-Δ strains are temperature sensitive due to lack of ac(4)C(12) and Um(44) and the consequent RTD of tRNA(Ser(CGA)) and tRNA(Ser(UGA)).

tRNAs marked with CCACCA are targeted for degradation.

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.

The yeast rapid tRNA decay pathway primarily monitors the structural integrity of the acceptor and T-stems of mature tRNA.

tRNAs, like other RNAs, are subject to quality control steps during and after biosynthesis. We previously described a rapid tRNA degradation (RTD) pathway in which the 5'-3' exonucleases Rat1 and Xrn1 degrade mature tRNA(Val(AAC)) in yeast mutants lacking m(7)G and m(5)C, and mature tRNA(Ser(CGA)) in mutants lacking Um and ac(4)C. To understand how the RTD pathway selects substrate tRNAs among different tRNAs lacking the same modifications, we used a genetic screen to examine tRNA(Ser(CGA)) variants.

Degradation of several hypomodified mature tRNA species in Saccharomyces cerevisiae is mediated by Met22 and the 5'-3' exonucleases Rat1 and Xrn1.

Mature tRNA is normally extensively modified and extremely stable. Recent evidence suggests that hypomodified mature tRNA in yeast can undergo a quality control check by a rapid tRNA decay (RTD) pathway, since mature tRNA(Val(AAC)) lacking 7-methylguanosine and 5-methylcytidine is rapidly degraded and deacylated at 37 degrees C in a trm8-Delta trm4-Delta strain, resulting in temperature-sensitive growth. We show here that components of this RTD pathway include the 5'-3' exonucleases Rat1 and Xrn1, and Met22, which likely acts indirectly through Rat1 and Xrn1.