Identification of SL addition trans-splicing acceptor sites in the internal transcribed spacer I region of pre-rRNA in Leishmania (Leishmania) amazonensis

Trypanosomatidae is a family of early branching eukaryotes harbouring a distinctive repertoire of gene expression strategies. Functional mature messenger RNA is generated via the trans-splicing and polyadenylation processing of constitutively transcribed polycistronic units. Recently, trans-splicing of pre-small subunit ribosomal RNA in the 5' external transcribed spacer region and of precursor tRNAsec have been described. Here, we used a previously validated semi-nested reverse transcription-polymerase chain reaction strategy to investigate internal transcribed spacer (ITS) I acceptor sites in total RNA from Leishmania (Leishmania) amazonensis. Two distinct spliced leader-containing RNAs were detected indicating that trans-splicing reactions occur at two AG acceptor sites mapped in this ITS region. These data provide further evidence of the wide spectrum of RNA molecules that act as trans-splicing acceptors in trypanosomatids.

RNA polymerase I promoter and splice acceptor site recognition affect gene expression in non-pathogenic Leishmania species

Leishmania (Sauroleishmania) tarentolae has biotechnological potential for use as live vaccine against visceral leishmaniasis and as a system for the over expression of eukaryotic proteins that possess accurate post-translational modifications. For both purposes, new systems for protein expression in this non-pathogenic protozoan are necessary. The ribosomal RNA promoter proved to be a stronger transcription driver since its use yielded increased levels of recombinant protein in organisms of both genera Trypanosoma or Leishmania. We have evaluated heterologous expression systems using vectors with two different polypyrimidine tracts in the splice acceptor site by measuring a reporter gene transcribed from L. tarentolae RNA polymerase I promoter. Our data indicate that the efficiency of chloramphenicol acetyl transferase expression changed drastically with homologous or heterologous sequences, depending on the polypyrimidine tract used in the construct and differences in size and/or distance from the AG dinucleotide. In relation to the promoter sequence the reporter expression was higher in heterologous lizard-infecting species than in the homologous L. tarentolae or in the mammalian-infecting L. (Leishmania) amazonensis.

Effect of splice-site polymorphisms of the TMPRSS4, NPHP4 and ORCTL4 genes on their mRNA expression.

Genetic polymorphisms associated with structural changes of their gene product are important in terms of their potential relation with diseases. Therefore, in this study, splice-site variants of the transmembrane serine protease gene TMPRSS4, nephronophthisis gene NPHP4, and organic-cation transporter gene ORCTL4, were selected from the dbSNP single nucleotide polymorphism database as candidates to identify genetic polymorphisms associated with a structural change in their mRNA transcripts. The allele frequencies of the TMPRSS4 c.4-7A>G, NPHP4 c.2818-2A>T, and ORCTL4 c.517-2A>C polymorphisms in a Japanese population were determined to be 0.42, 0.10, and 0.27, respectively, by PCR-SSCP analysis. Next, the effect of these polymorphisms on the mode of pre-mRNA splicing was investigated by RT-PCR analysis followed by sequencing analysis. The TMPRSS4, NPHP4, and ORCTL4 polymorphisms were associated with the production of the r.4-6_4-1ins transcript, the r.2818_2823del and r.2818_2859del transcripts, and the r.517-94_517-1ins; r.517-2a>c and r.517_620del transcripts, respectively. Since the proteins encoded by all these transcripts are associated with relatively significant structural changes in the form amino acid insertion/deletion and premature termination, their functional ability may be greatly reduced. Our demonstration of structural changes in mRNA transcripts as a result of splice-site polymorphisms implies that they may be of biological significance in certain pathological conditions.