Insect small nuclear RNA gene promoters evolve rapidly yet retain conserved features involved in determining promoter activity and RNA polymerase specificity

In animals, most small nuclear RNAs (snRNAs) are synthesized by RNA polymerase II (Pol II), but U6 snRNA is synthesized by RNA polymerase III (Pol III). In Drosophila melanogaster, the promoters for the Pol II-transcribed snRNA genes consist of approximately 21 bp PSEA and approximately 8 bp PSEB. U6 genes utilize a PSEA but have a TATA box instead of the PSEB. The PSEAs of the two classes of genes bind the same protein complex, DmSNAPc. However, the PSEAs that recruit Pol II and Pol III differ in sequence at a few nucleotide positions that play an important role in determining RNA polymerase specificity. We have now performed a bioinformatic analysis to examine the conservation and divergence of the snRNA gene promoter elements in other species of insects. The 5' half of the PSEA is well-conserved, but the 3' half is divergent. Moreover, within each species positions exist where the PSEAs of the Pol III-transcribed genes differ from those of the Pol II-transcribed genes. Interestingly, the specific positions vary among species. Nevertheless, we speculate that these nucleotide differences within the 3' half of the PSEA act similarly to induce conformational alterations in DNA-bound SNAPc that result in RNA polymerase specificity.     

Conserved sequences in the U2 snRNA-encoding genes of Kinetoplastida do not include the putative branchpoint recognition region

The U2 small nuclear RNA (snRNA) of Trypanosoma brucei gambiense, a flagellated protozoon of the order Kinetoplastida, is 148 nucleotides (nt) long, and thus the smallest U2 snRNA identified so far. To examine the evolutionary conservation of this RNA among Kinetoplastida, we have cloned and sequenced the U2 genes from Trypanosoma congolense and Leishmania mexicana amazonensis, which are 145 and 141 nt in length, respectively. The sequences of the Kinetoplastida U2 snRNAs are essentially identical in the 5' half of the molecule. Surprisingly, the putative branch site recognition sequence of L. m. amazonensis U2 snRNA shows two nt changes when compared with the other two U2 snRNAs. The sequence of the 3' half of the Kinetoplastida U2 snRNAs is less conserved with T. congolense and L. m. amazonensis RNAs showing 23 and 35 nt sequence variations, respectively, when compared with the corresponding sequence of the T. b. gambiense U2 snRNA. Alignment of the flanking regions of the U2 genes revealed several elements which are conserved both in sequence and in position relative to the U2 coding region and which may function in the biosynthesis of U2 snRNAs. One upstream element specifically binds protein factor(s) present in T. brucei nuclear extracts.     

Schizosaccharomyces U6 genes have a sequence within their introns that matches the B box consensus of tRNA internal promoters.

The gene for the U6 small nuclear RNA (snRNA) in the fission yeast Schizosaccharomyces pombe is interrupted by an intron whose structure is similar to those found in messenger RNA precursors (pre-mRNAs) (1). This is the only known example of a split snRNA gene from any organism--animal, plant, or yeast. To address the uniqueness of the S. pombe U6 gene, we have investigated the structures of the U6 genes from five Schizosaccharomyces strains and three other fungi. A fragment of the U6 coding sequence was amplified from the genomic DNA of each strain by the polymerase chain reaction (PCR). The sizes of the PCR products indicated that all of the fission yeast strains possess intron-containing U6 genes; whereas, the U6 genes from the other fungi appeared to be uninterrupted. The sequences of the Schizosaccharomyces U6 gene fragments revealed that each had an intron of approximately 50 base pairs in precisely the same position. In addition to the splice sites and putative branch point regions, a sequence immediately upstream of the branch point consensus was found to be conserved in all of the Schizosaccharomyces U6 genes. This sequence matches the consensus for the B box of eukaryotic tRNA promoters. These results raise the interesting possibility that synthesis of U6 RNA in fission yeast might involve the use of internal promoter elements similar to those found in other genes transcribed by RNA polymerase III.