TY - CHAP
T1 - Analysis of 3' End Modifications in microRNAs by High-Throughput Sequencing
AU - Reimão-Pinto, Madalena M
AU - Rodrigues-Viana, Angela M
AU - Ameres, Stefan L
N1 - Publisher Copyright:
© 2018, Springer Science+Business Media, LLC, part of Springer Nature.
PY - 2018
Y1 - 2018
N2 - MicroRNAs are ~22 nt small, non-coding RNAs that direct posttranscriptional silencing of gene expression to regulate animal development, physiology, and disease. An emerging mechanism that controls the biogenesis of microRNAs is the addition of non-templated nucleotides, predominantly uridine, to the 3' end of precursor-microRNAs, in a process that is commonly referred to as tailing. Here, we describe methods that enable the systematic characterization of tailing events in mature microRNAs and their precursors. We report protocols for untargeted and targeted cDNA library preparation procedures, as exemplified in the context of the model organism Drosophila melanogaster and focusing on precursor-microRNAs. We also refer to a dedicated computational framework for the subsequent analysis of untemplated nucleotide additions in cDNA libraries. The described methods for the systematic characterization of posttranscriptional modifications in gene regulatory small RNAs and their precursors will be instrumental in clarifying regulatory concepts that control posttranscriptional gene silencing.
AB - MicroRNAs are ~22 nt small, non-coding RNAs that direct posttranscriptional silencing of gene expression to regulate animal development, physiology, and disease. An emerging mechanism that controls the biogenesis of microRNAs is the addition of non-templated nucleotides, predominantly uridine, to the 3' end of precursor-microRNAs, in a process that is commonly referred to as tailing. Here, we describe methods that enable the systematic characterization of tailing events in mature microRNAs and their precursors. We report protocols for untargeted and targeted cDNA library preparation procedures, as exemplified in the context of the model organism Drosophila melanogaster and focusing on precursor-microRNAs. We also refer to a dedicated computational framework for the subsequent analysis of untemplated nucleotide additions in cDNA libraries. The described methods for the systematic characterization of posttranscriptional modifications in gene regulatory small RNAs and their precursors will be instrumental in clarifying regulatory concepts that control posttranscriptional gene silencing.
KW - Animals
KW - Drosophila melanogaster
KW - Gene Library
KW - Gene Silencing
KW - High-Throughput Nucleotide Sequencing/methods
KW - MicroRNAs/biosynthesis
KW - RNA Processing, Post-Transcriptional
KW - 3′ end tailing analysis
KW - Posttranscriptional RNA modifications
KW - Small RNA library preparation
KW - Targeted pre-miRNA sequencing
UR - http://www.scopus.com/inward/record.url?scp=85049366478&partnerID=8YFLogxK
U2 - 10.1007/978-1-4939-8624-8_10
DO - 10.1007/978-1-4939-8624-8_10
M3 - Chapter
C2 - 29959678
VL - 1823
T3 - Methods in Molecular Biology
SP - 115
EP - 139
BT - miRNA Biogenesis
ER -