Structure and expression of novel Caenorhabditis elegans splice leader (SL) RNA genes

Abstract

I have identified {dollar}\sim{dollar}30 novel SL RNA genes in the C. elegans genome. Five representative SL RNA genes have been cloned and sequenced. Three of the novel SL RNA genes (SL3{dollar}\alpha{dollar}, SL4 and SL5 RNA) are contained within a 3 kbp region of chromosome III. This locus was completely sequenced and the genomic organization of the SL RNA genes was determined. Inverted repeats of {dollar}\sim{dollar}150 nucleotides flank the SL RNA locus. Structures of the SL3{dollar}\beta{dollar} and SL3{dollar}\gamma{dollar} genes, which are located on chromosome II, were also determined. The SL RNA genes encode three distinct splice leader sequences (SL3-5). Each novel leader sequence is more closely related to SL2 than SL1. The 3{dollar}\sp\prime{dollar} end segments of the SL RNA transcripts contain sequence elements that are required for the trans-splicing reaction. 5{dollar}\sp\prime{dollar} flanking regions of all the SL RNA genes do not posses TATA boxes but do contain sequences that correspond to a proximal sequence element (PSE), which is a consensus promoter element initially identified in snRNA genes. Expression of novel SL RNAs was demonstrated by protecting full-length transcripts against RNAse digestion by hybridization with their corresponding antisense sequences (RNAse protection). This method was applied to measure the relative abundance of the SL RNA transcripts by quantifying the amount of protected RNA transcript in a fixed amount of total RNA. The five novel SL RNA transcripts, are approximately 1% as abundant as SL2{dollar}\alpha{dollar} RNA. However, collectively the total pool of novel SL sequences in poly A{dollar}\sp-{dollar} RNA (full length SL RNAs) and the amounts of novel RNA sequences transferred to mRNAs are similar to the levels of SL2 sequences in the poly A{dollar}\sp-{dollar} fractions of C. elegans RNA. I have also established that the novel splice leaders, found at the 5{dollar}\sp\prime{dollar} terminus of the novel SL RNAs, are utilized in in vivo trans-splicing reactions. This was accomplished by establishing RNAse protection conditions that detect mismatch in any nucleotide of the short splice leader sequence. The frequency of novel, SL1 and SL2 leader utilization in trans-splicing reactions was determined by quantitating the levels of the splice leaders that were protected in poly A{dollar}\sp+{dollar} RNA. Collectively, the novel SL sequences are used approximately {dollar}\sim{dollar}8% as frequently as the SL1 sequence. One of the novel leader sequences, SL4, is used 10% as frequently as SL2. Another novel leader sequence SL3, is used with the same frequency as SL2. This novel leader sequence appears on the 5{dollar}\sp\prime{dollar} end of tra-2 mRNA. Northern blot analysis demonstrated that a heterogeneous population of mRNAs are trans-spliced with novel SLs.;To examine the possibility that the choice of leader sequence may be developmentally regulated, the SL RNA content was measured in RNA isolated from developmentally staged worms. Most SL RNA levels remain constant throughout development. However, the SL4 RNA transcript increased significantly from the L1 to the adult stage. Transcriptional activity of the SL4 gene was studied by creating transgenic nematodes in which 800 bp of the SL4 promoter drives lacZ expression. (Abstract shortened by UMI.).