RNA trans-splicing in Fasciola hepatica. Identification of a spliced leader (SL) RNA and SL sequences on mRNAs.

R.E Davis,H Singh,C Botka,C Hardwick,M Ashraf El Meanawy,J Villanueva

doi:10.1016/s0021-9258(17)32122-1

R.E Davis, H Singh + Show 4 more

Open Access

https://doi.org/10.1016/s0021-9258(17)32122-1

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Abstract

We have identified a new spliced leader (SL) in Fasciola hepatica by characterizing the 5'-terminal sequences of its enolase mRNA, an mRNA also trans-spliced in the flatworm Schistosoma mansoni (Rajkovic, A., Davis, R.E., Simonsen, J.N., and Rottman, F.M. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 8879-8883). This 37-nucleotide spliced leader is at the 5' ends of multiple Fasciola mRNAs and is likely to be derived from the 5' terminus of a nonpolyadenylated, 108-nucleotide RNA with a trimethylguanosine cap. The SL RNA gene is present in approximately 100 copies within a 1.1-kilobase genomic tandem repeat. Secondary structure predictions indicate that the Fasciola SL RNA contains three stem loops in contrast to two previously observed in S. mansoni. Fasciola and S. mansoni SLs are likely to be evolutionarily related although their sequence identity is only 65%. In contrast with nematodes, absolute conservation of SL sequences and secondary structure does not occur in trematodes. A spliced leader in Fasciola indicates that trans-splicing is likely to be a common feature in other trematodes and perhaps other flatworms.

Highlights

We have identified a new spliced leader (SL)in Fas- and common form of geneexpression in early metazoa [8]
Fasciola mRNAs and is likely to be derived from the 5’ The sequence of the spliced leader in multicellular nematerminus of a nonpolyadenylated, 108-nucleotide RNA todes is absolutely conserved in all members of the phylum with a trimethylguanosinecap.The SL RNA gene is pres- examined
We describe and provide evidence for a Fasciola SL RNA and its secondary structure, the presence of the spliced leader on multiple mRNAs, Trans-splicing is an RNA processing event that accurately and theSL RNA gene contained within its genomic repeat

Summary

MATERIALS AND METHODS

One form of trans-splicing, a leader sequence PCR was performed using the anchor primer provided with the 5‘ RACE kit (with C-tailedcDNA) or 5”TCTAGAACTAGTGGATCCCCCCCCCCCCC-3’(with G-tailedcDNA) and theFasciola enolase-specificoligonucleotide, 5‘-CAUCAUCAUCAUATGCCT“TGGTCGATAGCTM-3‘ (U= deoxyuridine) (Fas.Eno.; see Fig). RNA and oligo(dT)were removed by RNase H treatment and GlassMax purification, and the cDNAs C-tailed as described in theLife Technologies 5' RACE kit. The cDNAs were amplified using the Life Technologies anchor primer and CAUCAUCAUCAU-oligo(dT),,using the conditions described above for5' RACE. Amplified cDNAs greater than 300 base pairs in size were cloned into pAMP vector [15].cDNAs with the Fasciola spliced leader sequence were identified bycolony hybridization with an end-labeled oligonucleotidecorrespondingto the complement of nucleotides of the spliced leader (Fas.Eno.4;Fig. 1). Stanford University) was screened with a random primerlabeled schistosome enolase cDNA2 as describedpreviously [10].An adult Xgtll cDNA library (generously provided by Allison Rice-Ficht, Texas A&M University) was screened with an oligonucleotide corresponding to the spliced leader (Fas.Eno.~)(10). Biochemical Corp.) as described [8,10]

RESULTS

B G A T CGBA T C

DISCUSSION

C C u u CA