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RNA structure, binding, and coordination in Arabidopsis

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From the moment of transcription, up through degradation, each RNA transcript is bound by an ever‐changing cohort of RNA binding proteins. The binding of these proteins is regulated by both the primary RNA sequence, as well as the intramolecular RNA folding, or secondary structure, of the transcript. Thus, RNA secondary structure regulates many post‐transcriptional processes. With the advent of next generation sequencing, several techniques have been developed to generate global landscapes of both RNA–protein interactions and RNA secondary structure. In this review, we describe the current state of the field detailing techniques to globally interrogate RNA secondary structure and/or RNA–protein interaction sites, as well as our current understanding of these features in the transcriptome of the model plant Arabidopsis thaliana. WIREs RNA 2017, 8:e1426. doi: 10.1002/wrna.1426

RNA binding proteins are essential for proper flower development in Arabidopsis. HLP1 binds to the distal polyadenylation signal (PAS) on the RNA binding protein, FCA, transcript, which produces the full‐length transcript. This full‐length transcript is then translated to produce FCAγ protein which then, along with another RNA binding protein, FPA, and a polyadenylation complex protein, FY, promotes the use of the proximal PAS on the FLC antisense transcripts. The choice of the proximal PAS triggers FLD‐dependent demethylation of H3K4me2, which, in turn, prevents transcription of the FLC gene, which results in proper flowering. When HLP1 is not present, it can no longer promote polyadenylation at the distal PAS of FCA, which leads to a generation of a truncated protein, FCAβ, which cannot bind and promote polyadenylation at the proximal PAS on FLC antisense transcripts. Therefore, FLD‐dependent demethylation of H3K4me2 does not occur and the FLC transcript is transcribed and translated, leading to delayed flowering.
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An overview of high‐throughput sequencing‐based RNA secondary structure probing techniques. (a)–(c) A representative hairpin loop is shown with double‐stranded (green) and single‐stranded (blue) regions. The nuclease‐based techniques cause cleavage between two adjacent nucleotides (triangles), which results in the sequencing reads shown below the hairpin loop for (a) FragSeq, (b) PARS, or (c) ds/ssRNA‐seq and PIP‐seq. (d)–(e) Chemical modifier‐based methods use a reagent to modify unpaired nucleotides in (d) DMS/structure‐seq and (e) icSHAPE.
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An overview of techniques used to interrogate in vitro, in vivo, and genome‐wide techniques used to study RNA–protein interactions. (a) and (b) In vitro techniques. (a) Electrophoretic mobility shift assays (EMSAs). (b) Systematic evolution of ligands by exponential enrichment (SELEX). (c)–(e) Target‐specific in vivo techniques. (c) RNA immunoprecipitation (RIP). RIP can be followed by RT‐qPCR (RIP‐qPCR), microarray (RIP‐chip), or RNA sequencing (RIP‐seq). (d) Crosslinking followed by Immunoprecipitation (CLIP). CLIP can be followed by RT‐qPCR (CLIP‐qPCR), microarray (CLIP‐chip), or RNA sequencing (CLIP‐seq). (e) Photoactivatable ribonucleoside enhanced crosslinking and immunoprecipitation (PAR‐CLIP). (f)–(g) Genome‐wide techniques. (f) Global photoactivatable ribonucleoside enhanced crosslinking and immunoprecipitation (gPAR‐CLIP). (g) Protein interaction profile sequencing (PIP‐seq).
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RNA binding proteins regulate alternative splicing of essential circadian rhythm genes. Proper circadian rhythm timing is dependent on alternative splicing of transcripts encoding specific components of the plant circadian clock. (a) Retention of the fourth intron in the CCA1 transcript results in an extended 5′ UTR (light blue boxes) and the use of a distal start codon. The resulting protein lacks the MYB domain, which is essential for the transcription factor activity of this protein, and cannot properly regulate circadian transcription. Thus, plants under high light and low temperature only express the CCA1β isoform of the protein, which results in plants with a shortened circadian period. (b) Proper splicing of circadian rhythm gene PRR9 is dependent on PRMT5 (green circle). Mutants lacking PRMT5 (prmt5 mutants) only have PRR9 transcripts with a retained third intron, resulting in a protein that is immediately degraded. Thus, prmt5 mutant plants display an elongated circadian period.
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RNA Structure and Dynamics > RNA Structure, Dynamics and Chemistry
RNA Interactions with Proteins and Other Molecules > Protein–RNA Recognition

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