Abstract
The nucleotides involved in RNA-RNA interaction can be tagged by chemical- or UV-induced crosslinking, and further identified by classical or modern high throughput techniques. The contacts of mRNA with 18S rRNA that occur along the mRNA channel of 40S subunit have been mapped by site-specific UV crosslinking followed by reverse transcriptase termination sites (RTTS) using radioactive or fluorescent oligonucleotides. However, the sensitivity of this technique is restricted to the detection of those fragments that resulted from the most frequent crosslinkings. Here, we combined RTTS with RNAseq to map the mRNA-18S rRNA contacts with a much deeper resolution. Although aimed to detect the interaction of mRNA with the ES6S region of 18S rRNA, this technique can also be applied to map the interaction of mRNA with other non-coding RNA molecules (e.g., snRNAs, microRNAs and lncRNAs) during transcription, splicing or RNA-mediated postranscriptional regulation.
Keywords: RNA-RNA interaction, Translation initiation, Ribosome, mRNA, Site-specific crosslinking, Reverse transcription stop, RNAseq
Background
Interaction of mRNA with non-coding RNAs is involved in every step of mRNA life cycle, from its biogenesis and processing into the nucleus to translation and ultimate degradation in the cytoplasm. These interactions can occur either within large molecular machines as ribosome and spliceosome, and in smaller complexes such as RISC (RNA-induced silencing complex) or during lncRNA-mediated gene expression regulation (Pisarev et al., 2008; Engreitz et al., 2014; Sharma et al., 2016). For translation, the use of in vitro reconstructed initiation complexes or drugs that freeze ribosomes during the initiation or elongation steps have allowed to snapshot those residues in mRNA and ribosomal RNAs involving in RNA-RNA contact. The incorporation of photoactivatable nucleotides (e.g., 4-thio-UTP) surrounding the AUGi in the mRNA allows site-specific crosslinking upon UV irradiation at 360 nm without affecting non-labeled residues. The resulting crosslinking adducts block primer extension by reverse transcriptase, thus allowing the identification of the crosslinking sites by sequencing the 3′ end of the resulting cDNA fragment (Kielpinski et al., 2013). Recently, a combination of reverse transcriptase termination sites (RTTS) with RNAseq has been described, allowing the identification of every premature stop during reverse transcriptase (Díaz-López et al., 2019). Apart from UV-induced crosslinking, there are other sources of RT stops, including accidental fragmentation of RNA template during extraction or preexisting chemical modification in mRNA residues (methylation and pseudourylation) that must be controlled during the experiment. Here, we describe in more detail this protocol that could be potentially applied to map any RNA-RNA interaction with nucleotide resolution using a labeled RNA bait. Our data working on mRNA-rRNA interaction during translation initiation indicates that the sensitivity of this protocol could be enough to detect even transient RNA-RNA interactions that regulate gene expression.
Materials and Reagents
Note: Make sure all reagents and materials are RNase-free. Materials are stored at room temperature unless otherwise indicated.
Equipment
Software
Procedure
Data analysis
FASTQ files from sequencing are processed under the FASTX software pipeline (http://hannonlab.cshl.edu/fastx_toolkit/galaxy.html):
Recipes
Acknowledgments
The technical support of Laura Barbado is acknowledged. This work was funded by Ministerio de Economía y Competitividad (BFU2017-84955-R) grant.
Competing interests
The authors declare neither conflicts of interest nor competing interests.
References
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