Spliced leader (SL) trans-splicing is a critical element of gene expression in a number of eukaryotic groups. This process is arguably best understood in nematodes, where biochemical and molecular studies in Caenorhabditis elegans and Ascaris suum have identified key steps and factors involved. Despite this, the precise details of SL trans-splicing have yet to be elucidated. In part, this is because the systematic identification of the molecules involved has not previously been possible due to the lack of a specific phenotype associated with defects in this process. We present here a novel GFP-based reporter assay that can monitor SL1 trans-splicing in living C. elegans. Using this assay, we have identified mutants in sna-1 that are defective in SL trans-splicing, and demonstrate that reducing function of SNA-1, SNA- 2 and SUT-1, proteins that associate with SL1 RNA and related SmY RNAs, impairs SL trans-splicing. We further demonstrate that the Sm proteins and pICln, SMN and Gemin5, which are involved in small nuclear ribonucleoprotein assembly, have an important role in SL trans-splicing. Taken together these results provide the first in vivo evidence for proteins involved in SL trans-splicing, and indicate that continuous replacement of SL ribonucleoproteins consumed during trans-splicing reactions is essential for effective trans-splicing.
Some strains were provided by the CGC, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440). We would also like to thank Prof. Shohei Mitani,at the National Bioresource Project for the Experimental Animal ‘Nematode C. elegans’, Japan, for FX3079. We are grateful to Prof. Tom Blumenthal (University of Colorado, Boulder) for suggestions and support of this work; and to Kathrine Wood for her contribution to the initial stages of part of this work. Author contributions. L.P., G.P., R.F., N.H., J.P. and B.M. performed experiments; B.M., J.P. and B.C. designed and lead the study; B.M. and J.P. drafted the manuscript. All authors reviewed the manuscript.
Biotechnology and Biological Sciences Research Council (BBSRC) [Project grant BB/J007137/1]; Medical Research Council (MRC) Confidence in Concept 2014 - University of Aberdeen Award(MC PC 14114v.2); University of Aberdeen Elphinstone Scholarship (to R.F.) and TET Fund support through Adekunle Ajasin University, Nigeria (to R.F.). Funding for open access charge: Biotechnology and Biological Sciences Research Council and Medical Research Council.
- rna splicing