Extensive variation in the 5′-UTR of Dicer mRNAs influences translational efficiency

doi:10.1016/j.bbrc.2005.07.138

Biochemical and Biophysical Research Communications

Volume 335, Issue 3, 30 September 2005, Pages 643-650

https://doi.org/10.1016/j.bbrc.2005.07.138 Get rights and content

Abstract

The Dicer enzyme is a key component of the RNA interference pathway and also responsible for the processing of micro RNAs, non-coding RNA molecules which regulate the activity of mRNAs by antisense base pairing. Little is known about the structure and regulation of human Dicer mRNA. A comprehensive characterization of Dicer 5′-untranslated region (5′-UTR) RNA structure revealed important diversity within human Dicer mRNA transcripts. Three exon 1 variants were defined, some of which exhibited very restricted patterns of tissue distribution. A number of alternatively spliced 5′-leader exons were also noted, revealing the potential for complex post-transcriptional regulation. Surprisingly, this diversity all occurred within the 5′-UTR of Dicer mRNAs and did not affect the coding region. The Dicer mRNA 5′-UTR variants had profound effects on translational efficiency both in vitro and in transiently transfected cells. A number of major Dicer RNA species are inefficient substrates for the translational machinery.

Section snippets

Materials and methods

cDNA and genomic characterization. Expressed sequence tag (EST) analyses, sequencing of IMAGE cDNA clones, RNA ligase mediated rapid amplification of 5′-cDNA ends, and genomic DNA analysis were utilized to provide evidence for 5′-UTR diversity in Dicer mRNA transcripts. Human genomic sequences for Dicer were obtained from bacterial artificial chromosome C-2240H23 of library Caltech-D from chromosome 14. 5′-RACE was performed with total cellular RNA derived from normal human tissues: brain,

Cloning of multiple exon 1 variants, alternatively spliced exons, and their genomic organization

5′-RACE was utilized to isolate 5′-leader sequences upstream of exon 2, which contains the translational initiation codon. A total of 94 RACE clones derived from human brain, testes, lung, and vascular endothelial cells were analyzed. Several distinct cDNA 5′-termini were identified. Sequence diversity was located upstream of exon 2. Stop codons were evident in all three translation reading frames immediately upstream of the initiating AUG in exon 2, thus all newly identified sequences

Discussion

Dicer expression is regulated by multiple processes including transcription, pre-mRNA splicing, and translation. Tissue-specific expression of Dicer mRNA variants was observed to result from the differential usage of three distinct promoters. Additional mechanisms were also superimposed upon this transcriptional diversity: three alternatively spliced 5′-UTR exons, varied transcriptional start sites, and the usage of alternative intron donor sites. Importantly, this diversity did not affect the

Acknowledgments

P.A.M. is a recipient of a Heart and Stroke Foundation of Canada Career Investigator Award and supported by Operating Grant T-5003 from the Heart and Stroke Foundation of Canada. S.S. is a recipient of a Natural Sciences and Engineering Research Council of Canada Post-Graduate Scholarship.

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^☆: Abbreviations: 5′-UTR, 5′-untranslated region; miRNA, micro RNA; ORF, open reading frame; siRNA, small interfering RNA; RNAi, RNA interference; RPA, RNase protection assay.

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Extensive variation in the 5′-UTR of Dicer mRNAs influences translational efficiency☆

Abstract

Section snippets

Materials and methods

Cloning of multiple exon 1 variants, alternatively spliced exons, and their genomic organization

Discussion

Acknowledgments

Curr. Opin. Biotechnol.

Cell

Cell

J. Biol. Chem.

J. Biol. Chem.

Lancet

Curr. Biol.

Dev. Biol.

Cell

Dicer is essential for mouse development

Nat. Genet.

The microRNA-producing enzyme Dicer1 is essential for zebrafish development

Nat. Genet.

Interferon antagonist proteins of influenza and vaccinia viruses are suppressors of RNA silencing

Proc. Natl. Acad. Sci. USA

Specific interference with gene expression induced by long, double-stranded RNA in mouse embryonal teratocarcinoma cell lines

Proc. Natl. Acad. Sci. USA

A role for the RNase III enzyme DCR-1 in RNA interference and germ line development in Caenorhabditis elegans

Science

Dicer functions in RNA interference and in synthesis of small RNA involved in developmental timing in C. elegans

Genes Dev.