Abstract
BackgroundThe Cleavage Stimulation Factor (CstF) is a required protein complex for eukaryotic mRNA 3'-processing. CstF interacts with 3'-processing downstream elements (DSEs) through its 64-kDa subunit, CstF-64; however, the exact nature of this interaction has remained unclear. We used EST-to-genome alignments to identify and extract large sets of putative 3'-processing sites for mRNA from ten metazoan species, including Homo sapiens, Canis familiaris, Rattus norvegicus, Mus musculus, Gallus gallus, Danio rerio, Takifugu rubripes, Drosophila melanogaster, Anopheles gambiae, and Caenorhabditis elegans. In order to further delineate the details of the mRNA-protein interaction, we obtained and multiply aligned CstF-64 protein sequences from the same species.ResultsWe characterized the sequence content and specific positioning of putative DSEs across the range of organisms studied. Our analysis characterized the downstream element (DSE) as two distinct parts – a proximal UG-rich element and a distal U-rich element. We find that while the U-rich element is largely conserved in all of the organisms studied, the UG-rich element is not. Multiple alignment of the CstF-64 RNA recognition motif revealed that, while it is highly conserved throughout metazoans, we can identify amino acid changes that correlate with observed variation in the sequence content and positioning of the DSEs.ConclusionOur analysis confirms the early reports of separate U- and UG-rich DSEs. The correlated variations in protein sequence and mRNA binding sequences provide novel insights into the interactions between the precursor mRNA and the 3'-processing machinery.
Highlights
The Cleavage Stimulation Factor (CstF) is a required protein complex for eukaryotic mRNA 3'-processing
While it is possible that the U/UG-rich downstream elements (DSEs) comprise one motif, several aspects of our analysis lead us to the conclusion that our results are consistent with the presence of distinct UG- and U-rich elements as proposed by McDevitt et al [9] and Gil and Proudfoot [10]. (This notably excludes C. elegans, which has no evidence of a UG-rich component.) While the positioning distributions of the U- and UG-rich sequences have considerable overlap, it is clear from Figures 2 and 3 that they are distinct
If the model we propose is accurate, and CstF-64 is responsible for interactions with both DSEs, it provides an explanation for the discrepancy between sequence
Summary
The Cleavage Stimulation Factor (CstF) is a required protein complex for eukaryotic mRNA 3'-processing. We used EST-to-genome alignments to identify and extract large sets of putative 3'processing sites for mRNA from ten metazoan species, including Homo sapiens, Canis familiaris, Rattus norvegicus, Mus musculus, Gallus gallus, Danio rerio, Takifugu rubripes, Drosophila melanogaster, Anopheles gambiae, and Caenorhabditis elegans. Cleavage and polyadenylation (3'-processing) are essential steps in eukaryotic mRNA formation that can effect transcript stability and function [1]. Selection of the 3'-processing site is directed by interactions between the polyadenylation machinery and cis-acting elements found both upstream and downstream of the 3'-processing site. The principle upstream cis-acting element is the highly conserved AAUAAA hexamer, which interacts with Cleavage and Polyadenylation Specificity Factor (CPSF) and is found in the majority of metazoan transcripts [1,3].
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