Synthesis of modified nucleotide polymers by the poly(U) polymerase Cid1: application to direct RNA sequencing on nanopores
Autor: | Mark Akeson, Jen Quick-Cleveland, Jenny Mai Vo, Logan Mulroney, Miten Jain, Manuel Ares |
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Rok vydání: | 2021 |
Předmět: |
Polymers
1.1 Normal biological development and functioning Saccharomyces cerevisiae RNA polymerase II Nanopores Underpinning research Schizosaccharomyces Genetics medicine Nucleotide Inosine Molecular Biology Polymerase chemistry.chemical_classification biology Sequence Analysis RNA Nucleotides Human Genome Polynucleotide Adenylyltransferase RNA biology.organism_classification Nucleotidyltransferases Biochemistry chemistry Schizosaccharomyces pombe biology.protein Nucleic acid Schizosaccharomyces pombe Proteins Generic health relevance Biochemistry and Cell Biology Sequence Analysis Developmental Biology medicine.drug |
Zdroj: | RNA (New York, N.Y.), vol 27, iss 12 |
ISSN: | 1469-9001 1355-8382 |
Popis: | Understanding transcriptomes requires documenting the structures, modifications, and abundances of RNAs as well as their proximity to other molecules. The methods that make this possible depend critically on enzymes (including mutant derivatives) that act on nucleic acids for capturing and sequencing RNA. We tested two 3’ nucleotidyl transferases, Saccharomyces cerevisiae poly(A) polymerase and Schizosaccharomyces pombe Cid1, for the ability to add base and sugar modified rNTPs to free RNA 3’ ends, eventually focusing on Cid1. Although unable to polymerize ΨTP or 1meΨTP, Cid1 can use 5meUTP and 4thioUTP. Surprisingly, Cid1 can use inosine triphosphate to add poly(I) to the 3’ ends of a wide variety of RNA molecules. Most poly(A) mRNAs efficiently acquire a uniform tract of about 50 inosine residues from Cid1, whereas non-poly(A) RNAs acquire longer, more heterogeneous tails. Here we test these activities for use in direct RNA sequencing on nanopores, and find that Cid1-mediated poly(I)-tailing permits detection and quantification of both mRNAs and non-poly(A) RNAs simultaneously, as well as enabling the analysis of nascent RNAs associated with RNA polymerase II. Poly(I) produces a different current trace than poly(A), enabling recognition of native RNA 3’ end sequence lost by in vitro poly(A) addition. Addition of poly(I) by Cid1 offers a broadly useful alternative to poly(A) capture for direct RNA sequencing on nanopores. |
Databáze: | OpenAIRE |
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