An experimental and genomic approach to the regulation of alternative pre-mRNA splicing in Drosophila rnp-4f
rnp-4f , a Drosophila gene containing nine spliceosomal introns, encodes a nuclear PRP protein that functions constitutively in spliceosome assembly. Nothing is known about molecular mechanisms regulating splicing decisions for this gene, or the factors that modulate the degree of mRNA translation for the encoded spliceosomal assembly factor during development. Here we report results of a systematic study of alternative pre-mRNA splicing in rnp-4f. Reverse transcription-polymerase chain reaction (RT-PCR) and hybridization analysis show that introns #I - IV and #VI - VIII are constitutively spliced, while five patterns of alternative splicing are observed in two other pre-mRNA regions. Intron V is infrequently spliced in all developmental stages, which results in generation of an in-frame stop codon and a predicted truncated protein lacking a nuclear localization signal, so that facultative splicing regulates the subcellular localization of the encoded protein. Intron 0 plus a portion of exon 2 (intron-0-alt) is alternatively spliced from the 5'-UTR, resulting in loss of an evolutionarily conserved stem-loop located in exon 2. Northern analysis of poly (A+) mRNAs reveals two differently sized rnp-4f mRNA isomers, one dominating during mid-embryo stages of CNS development. The size of one of these mRNAs corresponds to that predicted for the constitutively spliced rnp-4f transcript. The other mRNA corresponds to the predicted size of the alternatively spliced transcript in which intron 0 plus a portion of exon 2 is excised, a result confirmed by hybridization. Potential molecular mechanisms which could regulate the observed splicing patterns are discussed, specifically from the viewpoint of putative cis-regulatory elements within rnp-4f pre-mRNAs. The alternative mRNA isoforms observed within the 5'-UTR are predicted to affect regulatory processes, including translational efficiency. A model is proposed in which RNP-4F controls its own level of expression by feedback against evolutionarily conserved twin stem-loops located at the 3'-end of the intron-0-alt pre-mRNA tract.
School Location:USA - Ohio
Source Type:Master's Thesis
Keywords:alternative pre mrna splicing 5 utr regulation facultative intron cis regulatory elements stem loop models
Date of Publication:01/01/2005