Proteomics

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Uncovering deeply conserved motif combinations in rapidly evolving noncoding sequences


ABSTRACT: Here we focus on identifying conserved elements that are required for Chaserr function, a lncRNA that we recently characterized as being essential for mouse viability (https://doi.org/10.1038/s41467-019-13075-8). Chaserr is highly conserved in different species, and is located in close proximity (<2kb) to the transcription start site of CHD2. It acts in concert with the CHD2 protein to maintain proper Chd2 expression levels. Using the LncLOOM framework we identified AUGG enriched conserved elements which are located in the last exon of mouse Chaserr. In order to identify proteins that potentially bind to these conserved regions, we used in vitro transcription to generate the following biotinylated RNAs; along with their antisense controls: 1) The WT sequence, containing the first 322 nt of the last exon of Chaserr (bases 764–1086 of the NR_037601 isoform). An antisense control fragment was also prepared, labelled as AWT 2) The mutant conserved (MC) sequence, that contained AUGG→UACC mutations in four conserved motifs that were found using LncLOOM. An antisense control fragment was also prepared, labelled as AMC 3) The muatant all (MA) sequence in which all seven of the AUGG sites in the last exon were mutated to UACC. An antisense control fragment was also prepared, labelled as AMA 4) We also included a No Probe Control, which did not contain an RNA fragment. The fragments were incubated with N2a cell lysates followed by RNA-pulldown and Mass Spec analysis. The experiment was performed in triplicate. A total of 938 proteins were found to associate with the WT sequence: 74 of these were enriched ≥3-fold compared to the antisense sequence.We identified 9 proteins that had ≥2-fold higher recovery when using the WT sequence compared to both mutants. Included in these proteins was DHX36, which showed the highest enrichment compared to the mutated sequences. We validated the association of DHX36 with Chaserr through RNA immunoprecipitation experiments.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Mus Musculus (mouse)

SUBMITTER: Caroline Ross  

LAB HEAD: Igor Ulitsky

PROVIDER: PXD023093 | Pride | 2021-01-18

REPOSITORIES: Pride

Dataset's files

Source:
Action DRS
HF1_11249_IUL_1_AMA_MS_050520.raw Raw
HF1_11249_IUL_1_AMC_MS_050520.raw Raw
HF1_11249_IUL_1_AWT_MS_050520.raw Raw
HF1_11249_IUL_1_MA_MS_050520.raw Raw
HF1_11249_IUL_1_MC_MS_050520.raw Raw
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Publications

Uncovering deeply conserved motif combinations in rapidly evolving noncoding sequences.

Ross Caroline Jane CJ   Rom Aviv A   Spinrad Amit A   Gelbard-Solodkin Dikla D   Degani Neta N   Ulitsky Igor I  

Genome biology 20210111 1


<h4>Background</h4>Animal genomes contain thousands of long noncoding RNA (lncRNA) genes, a growing subset of which are thought to be functionally important. This functionality is often mediated by short sequence elements scattered throughout the RNA sequence that correspond to binding sites for small RNAs and RNA binding proteins. Throughout vertebrate evolution, the sequences of lncRNA genes changed extensively, so that it is often impossible to obtain significant alignments between sequences  ...[more]

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