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Identification of mRNA Localization Motifs through Cell Fractionation and Alternative Splicing Analysis
Author(s) -
Taliaferro J,
Vidaki Marina,
Gertler Frank,
Burge Christopher
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.562.30
Subject(s) - alternative splicing , rna splicing , polyadenylation , exon , biology , rna binding protein , subcellular localization , messenger rna , rna , gene isoform , gene knockdown , microbiology and biotechnology , gene expression , gene , genetics , cytoplasm
The biogenesis of mammalian mRNAs involves several regulated steps, including alternative splicing and alternative cleavage and polyadenylation. In addition, certain mRNAs can be localized to distinct regions of the cell, contributing to protein localization and complex formation. This is particularly important in polar cell types, such as neurons. Although several hundred mRNAs are present in neuronal projections, only a handful of RNA sequences are known to target an mRNA to a particular location. Using mechanical subcellular fractionation, RNA‐seq, and alternative splicing analysis, we have identified hundreds of mRNA regions associated with localization to neuronal projections in neuroblastoma cells and primary cortical neurons. These regions are overwhelmingly found in 3′ UTRs, and in particular are associated with alternative last exon splicing events and result from a characteristic alternative isoform organization. Localization‐associated 3′ UTRs are shorter, more conserved, more gene‐distal, and contain a higher propensity for secondary structure than other 3′ UTRs. Furthermore, they are sufficient to direct reporter RNA into neuronal projections and are enriched in binding sites for trans‐factors known to be involved in RNA localization. Knockdown of expression of these factors inhibits the localization of many transcripts. These data demonstrate the tight interplay between each step in gene expression as specific alternative splicing decisions in the nucleus determine the cytoplasmic localization fate of the transcript.