Abstract
Post-transcriptional regulatory networks are dependent on the interplay of many RNA-binding proteins having a major role in mRNA processing events in mammals. We have been interested in the concerted action of the two RNA-binding proteins hnRNP A1 and HuR, both stable components of immunoselected hnRNP complexes and having a major nuclear localization. Specifically, we present here the application of the RNA-immunoprecipitation (RIP)-Chip technology to identify a population of nuclear transcripts associated with hnRNP A1-RNPs as isolated from the nuclear extract of either HuR WT or HuR-depleted (KO) mouse embryonic fibroblast (MEF) cells. The outcome of this analysis was a list of target genes regulated via HuR for their association (either increased or reduced) with the nuclear hnRNP A1-RNP complexes. Real time PCR analysis was applied to validate a selected number of nuclear mRNA transcripts, as well as to identify pre-spliced transcripts (in addition to their mature mRNA counterpart) within the isolated nuclear hnRNP A1-RNPs. The differentially enriched mRNAs were found to belong to GO categories relevant to biological processes anticipated for hnRNP A1 and HuR (such as transport, transcription, translation, apoptosis and cell cycle) indicating their concerted function in mRNA metabolism.
Highlights
RNA-binding proteins (RBPs) that associate with RNA pol II transcriptsconstitute a large group of cellular proteins that are key components of macromolecular assemblies functioning in post-transcriptional events such as splicing, polyadenylation, transport, localization and stability/translation of mRNA
The general strategy applied in the present study was based on our previous identification of Heterogeneous nuclear ribonucleoproteins (hnRNPs) A1 as associating with HuR [31,32] and we focused on the identification, amongst the plethora of reported HuR targets, of those that were in stable association with hnRNP A1-RNP complexes pre-existing in the nuclear extracts of mammalian cell origin
mouse embryonic fibroblast (MEF) cells either HuR Wild Type (WT) or HuR KO, provided a suitable experimental system to compare within purified nuclear hnRNP complexes any co-selected RNA species
Summary
Constitute a large group of cellular proteins that are key components of macromolecular assemblies functioning in post-transcriptional events such as splicing, polyadenylation, transport, localization and stability/translation of mRNA. This is brought about by the extensive interplay amongst discrete sets of RBPs and their associated (pre)- and mRNA-target molecules in the form of ribonucleoprotein (RNP). The hnRNP proteins comprise a group of over 20 polypeptides in the range of 32 to 110 kDa and designated in order of increasing molecular weight as hnRNP A-U [8,9] They are abundant nuclear proteins containing at least one motif for binding to RNA (the RBD/RRM, KH or RGG domains)
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