TY - JOUR
T1 - Urb-RIP - An adaptable and efficient approach for immunoprecipitation of RNAs and associated RNAs/proteins
AU - Cottrell, Kyle A.
AU - Djuranovic, Sergej
N1 - Funding Information:
We thank the Kathleen Hall (Washington University Medical School) for a plasmid containing Urb-RRM1 and for advice. We also thank Denise Rogers and Yansel Nunez for assistance in cloning. This work was supported by an NIH training grant T32 GM007067 to KAC and NIH Grant RO1 GM112824 to SD.
Publisher Copyright:
© 2016 Cottrell, Djuranovic.This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
PY - 2016/12
Y1 - 2016/12
N2 - Post-transcriptional regulation of gene expression is an important process that is mediated by interactions between mRNAs and RNA binding proteins (RBP), non-coding RNAs (ncRNA) or ribonucleoproteins (RNP). Key to the study of post-transcriptional regulation of mRNAs and the function of ncRNAs such as long non-coding RNAs (lncRNAs) is an understanding of what factors are interacting with these transcripts. While several techniques exist for the enrichment of a transcript whether it is an mRNA or an ncRNA, many of these techniques are cumbersome or limited in their application. Here we present a novel method for the immunoprecipitation of mRNAs and ncRNAs, Urb - RNA immunoprecipitation (Urb-RIP). This method employs the RRM1 domain of the "resurrected" snRNA-binding protein Urb to enrich messages containing a stem-loop tag. Unlike techniques which employ the MS2 protein, which require large repeats of the MS2 binding element, Urb-RIP requires only one stem-loop. This method routinely provides over ∼100-fold enrichment of tagged messages. Using this technique we have shown enrichment of tagged mRNAs and lncRNAs as well as miRNAs and RNA-binding proteins bound to those messages. We have confirmed, using Urb-RIP, interaction between RNA PolIII transcribed lncRNA BC200 and polyA binding protein.
AB - Post-transcriptional regulation of gene expression is an important process that is mediated by interactions between mRNAs and RNA binding proteins (RBP), non-coding RNAs (ncRNA) or ribonucleoproteins (RNP). Key to the study of post-transcriptional regulation of mRNAs and the function of ncRNAs such as long non-coding RNAs (lncRNAs) is an understanding of what factors are interacting with these transcripts. While several techniques exist for the enrichment of a transcript whether it is an mRNA or an ncRNA, many of these techniques are cumbersome or limited in their application. Here we present a novel method for the immunoprecipitation of mRNAs and ncRNAs, Urb - RNA immunoprecipitation (Urb-RIP). This method employs the RRM1 domain of the "resurrected" snRNA-binding protein Urb to enrich messages containing a stem-loop tag. Unlike techniques which employ the MS2 protein, which require large repeats of the MS2 binding element, Urb-RIP requires only one stem-loop. This method routinely provides over ∼100-fold enrichment of tagged messages. Using this technique we have shown enrichment of tagged mRNAs and lncRNAs as well as miRNAs and RNA-binding proteins bound to those messages. We have confirmed, using Urb-RIP, interaction between RNA PolIII transcribed lncRNA BC200 and polyA binding protein.
UR - http://www.scopus.com/inward/record.url?scp=85003021443&partnerID=8YFLogxK
U2 - 10.1371/journal.pone.0167877
DO - 10.1371/journal.pone.0167877
M3 - Article
C2 - 27930710
AN - SCOPUS:85003021443
VL - 11
JO - PLoS ONE
JF - PLoS ONE
SN - 1932-6203
IS - 12
M1 - e0167877
ER -