Previous genetic and biochemical experiments have shown that the vaccinia virus J3 protein has three different roles in mRNA synthesis and modification. First, J3 is a (nucleoside-2'-O-)methyltransferase which methylates the 2' position of the first transcribed nucleotide, thus converting a cap-0 to a cap-1 structure at the 5' ends of mRNAs. Second, J3 is a processivity factor for the virus coded poly(A) polymerase. Third, J3 has recently been shown to have intermediate and late gene positive transcription elongation factor activity in vivo. Previous experiments have shown that the poly(A) polymerase stimulatory activity and the (nucleoside-2'-O-)methyltransferase activity are two independent functions of the protein that can be genetically separated through site-directed mutagenesis. In this article, the relationship between the J3-mediated transcription elongation activity and the two other functions of the protein was investigated by constructing several site-directed mutant viruses that contain specific defects in either methyltransferase or poly(A) polymerase processivity functions. The results demonstrate that the J3 positive transcription elongation factor activity is a third independent function of the protein that is genetically separable from its two other functions in mRNA modification. The results also show that neither the poly(A) polymerase stimulatory nor the methyltransferase activities of the J3 protein is essential for virus growth in cell culture.
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http://dx.doi.org/10.1006/viro.2002.1538 | DOI Listing |
Front Parasitol
January 2024
Department of Biomedical Sciences, University of Minnesota Medical School, Duluth, MN, United States.
RNA-specific nucleotidyltransferases (rNTrs) add nontemplated nucleotides to the 3 end of RNA. Two noncanonical rNTRs that are thought to be poly(A) polymerases (PAPs) have been identified in the mitochondria of trypanosomes - KPAP1 and KPAP2. KPAP1 is the primary polymerase that adds adenines (As) to trypanosome mitochondrial mRNA 3 tails, while KPAP2 is a non-essential putative polymerase whose role in the mitochondria is ambiguous.
View Article and Find Full Text PDFCommun Biol
January 2025
Department of Chemistry, Merkert Chemistry Center, Boston College, Chestnut Hill, MA, USA.
Pseudouridine (Ψ) is an abundant RNA chemical modification that plays critical biological functions. Current Ψ detection methods are limited in identifying Ψs at base-resolution in U-rich sequence contexts, where Ψ occurs frequently. Here we report "Mut-Ψ-seq" that utilizes the classic N-cyclohexyl N'-(2-morpholinoethyl)carbodiimide (CMC) agent and an evolved reverse transcriptase ("RT-1306") for Ψ mapping at base-resolution.
View Article and Find Full Text PDFSci Data
January 2025
Laboratory of RNA Biology, International Institute of Molecular and Cell Biology, Warsaw, 02-109, Poland.
Gametogenesis is a process in which dysfunctions lead to infertility, a growing health and social problem worldwide. In both spermatogenesis and oogenesis, post-transcriptional gene expression regulation is crucial. Essentially, all mRNAs possess non-templated poly(A) tails, whose composition and dynamics (elongation, shortening, and modifications) determine the fate of mRNA.
View Article and Find Full Text PDFArch Virol
December 2024
Key Laboratory for Agricultural Bioaffiliationersity for Pest Management of China, Ministry of Education, Yunnan Agricultural University, NO. 95, Jinhei Road, Panlong District, Kunming, Yunnan, 650201, China.
BMC Biol
December 2024
Life Science Research Centre, Faculty of Science, University of Ostrava, Ostrava, 710 00, Czech Republic.
Background: In trypanosomatids, a group of unicellular eukaryotes that includes numerous important human parasites, cis-splicing has been previously reported for only two genes: a poly(A) polymerase and an RNA helicase. Conversely, trans-splicing, which involves the attachment of a spliced leader sequence, is observed for nearly every protein-coding transcript. So far, our understanding of splicing in this protistan group has stemmed from the analysis of only a few medically relevant species.
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