Abstract
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.
Highlights
Regulation of gene expression at the post-transcriptional level is a complex process involving many trans factors such as RNA-binding proteins (RBPs), non-coding RNA, and ribonucleoproteins (RNPs) [1, 2]
We have incorporated a restriction enzyme site on one side of the stem-loop II (SLII)-tag to aid in subsequent cloning
Proteinase K treatment is an important step in the procedure much like in HITS-CLIP as it degrades proteins covalently bound to the RNA that could interfere with reverse transcriptase during cDNA synthesis [28]
Summary
Regulation of gene expression at the post-transcriptional level is a complex process involving many trans factors such as RNA-binding proteins (RBPs), non-coding RNA (ncRNA), and ribonucleoproteins (RNPs) [1, 2]. In order to fully understand this process for any given RNA it is essential that we know what factors are bound to the transcript. This knowledge will prove useful in designing therapies that target trans factors or the RNA itself. Current techniques for RNA purification fall into one of three classes: RBP-mediated [3,4,5,6,7,8,9,10], PLOS ONE | DOI:10.1371/journal.pone.0167877 December 8, 2016
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