Abstract
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.
Highlights
A wide range of eukaryotes engage in the trans-splicing of their pre-mRNAs, a process which results in the replacement of the 5 -end of the transcript with a short, ‘spliced leader’ (SL) [1,2]
We show that depletion of Sm protein expression, and factors involved in small nuclear RNPs (snRNPs) assembly, leads to reduced SL trans-splicing
To determine whether factors involved in snRNP assembly play a role in SL trans-splicing, we knocked down the expression of smn-1, smi-1, icln-1 by RNA interference (RNAi). smn-1 encodes a homolog of human SMN and smi-1 an ortholog of human Gemin2 that interacts with SMN, and, together with SMN, is a component of the SMN complex [45]
Summary
A wide range of eukaryotes engage in the trans-splicing of their pre-mRNAs, a process which results in the replacement of the 5 -end of the transcript with a short, ‘spliced leader’ (SL) [1,2]. To test the response of the transgenic strain to loss of SL trans-splicing, we carried out RNA interference (RNAi) targeted against sna-1 and sna-2, since these genes encode known components of the SL RNP, and sut-1, because of its genetic interaction with sna-1 and proposed role in Sm protein recycling [15].
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