A Candidate U1 Small Nuclear RNA for Trypanosomatid Protozoa

Murray N Schnare,Michael W Gray

doi:10.1074/jbc.274.34.23691

Abstract

In trypanosomatid protozoa, all mRNAs obtain identical 5'-ends by trans-splicing of the 5'-terminal 39 nucleotides of a small spliced leader RNA to appropriate acceptor sites in pre-mRNA. Although this process involves spliceosomal small nuclear (sn) RNAs, it is thought that trypanosomatids do not contain a homolog of the cis-spliceosomal U1 snRNA. We show here that a trypanosomatid protozoon, Crithidia fasciculata, contains a novel small RNA that displays several features characteristic of a U1 snRNA, including (i) a methylguanosine cap and additional 5'-terminal modifications, (ii) a potential binding site for common core proteins that are present in other trans-spliceosomal ribonucleoproteins, (iii) a U1-like 5'-terminal sequence, and (iv) a U1-like stem/loop I structure. Because trypanosomatid pre-mRNAs do not appear to contain cis-spliced introns, we argue that this previously unrecognized RNA species is a good candidate to be a trans-spliceosomal U1 snRNA.

Highlights

In trypanosomatid protozoa, all mRNAs have identical 5Јterminal sequences
Covery of a U1 snRNA homolog in Euglena gracilis [8], an organism that is related to trypanosomatid protozoa [9], prompted us to re-evaluate a possible role for U1 snRNA in trypanosomatids
This view is supported by a study in which a trypanosomatid (Leptomonas collosoma) spliced leader (SL) RNA sequence was placed upstream of a 3Ј-splice site, with the resulting chimeric substrate being efficiently spliced in a HeLa cell nuclear extract even after the 5Ј-end of Ͼ99% of the endogenous U1 snRNA had been removed by oligonucleotide-directed RNase H cleavage [7]

Summary

A Candidate U1 Small Nuclear RNA for Trypanosomatid Protozoa*

All mRNAs obtain identical 5؅-ends by trans-splicing of the 5؅-terminal 39 nucleotides of a small spliced leader RNA to appropriate acceptor sites in pre-mRNA This process involves spliceosomal small nuclear (sn) RNAs, it is thought that trypanosomatids do not contain a homolog of the cis-spliceosomal U1 snRNA. This view is supported by a study in which a trypanosomatid (Leptomonas collosoma) SL RNA sequence was placed upstream of a 3Ј-splice site, with the resulting chimeric substrate being efficiently spliced in a HeLa cell nuclear extract even after the 5Ј-end of Ͼ99% of the endogenous U1 snRNA had been removed by oligonucleotide-directed RNase H cleavage [7] This result provided support for an earlier proposal that U1 snRNA-like base pairing could be supplied by a region of the SL RNA upstream of the 5Ј-splice site [6]. We show that a representative trypanosomatid, Crithidia fasciculata, contains a novel small RNA that displays several characteristic features expected of a U1 snRNA homolog

EXPERIMENTAL PROCEDURES

A Candidate U1 snRNA for Trypanosomatid Protozoa

RESULTS AND DISCUSSION