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. 2017 Aug 21;45(14):8474-8483.
doi: 10.1093/nar/gkx500.

An in vivo genetic screen for genes involved in spliced leader trans-splicing indicates a crucial role for continuous de novo spliced leader RNP assembly

Lucas Philippe  1 , George C Pandarakalam  1 , Rotimi Fasimoye  1 , Neale Harrison  1 , Bernadette Connolly  1 , Jonathan Pettitt  1 , Berndt Müller  1
Affiliations

An in vivo genetic screen for genes involved in spliced leader trans-splicing indicates a crucial role for continuous de novo spliced leader RNP assembly

Lucas Philippe et al. Nucleic Acids Res. .

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.

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Figures

Figure 1.
Figure 1.
A novel reporter gene assay for the in vivo detection of reduced SL1 trans-splicing. (A) The Pvit-2::outron::gfpM1A transgene engineered to monitor SL1 trans-splicing. The original ATG of the GFP open reading frame (yellow shading) was changed to a GCT alanine codon. Note the gfp gene contains three introns. The two mRNA products produced in the presence, or absence of SL1 trans-splicing are shown below, and a cartoon of the predicted GFP expression in wild-type (wt) worms (green shading indicates intestinal GFP fluorescence). (B) Detection of SL1 trans-splicing inhibition by fluorescence microscopy. Micrographs of GFP expression in animals carrying the Pvit-2::outron::GFPM1A transgene subjected to sna-1, sna-2, sut-1 or unc-22(RNAi). Exposure times were identical for all micrographs (5 ms). The scale bar corresponds to 100 μm. (C and D) Quantitation of GFP expression in animals carrying the Pvit-2::outron::GFPM1A transgene subjected to sna-1, sna-2, sut-1 or unc-22(RNAi). (C) The proportion of GFP fluorescent animals was determined by fluorescence microscopy. Error bars represent the standard deviation based on between two and five independent experiments (unc-22(RNAi): 2; sna-1(RNAi), sna-2(RNAi): 3; sut-1(RNAi): 5). The total number of animals analyzed is indicated at the top of the graph. (D) The intensity of GFP fluorescence (in arbitrary units) was determined from micrographs as shown in (B), and standardized with respect to mCherry fluorescence. The graph plots the GFP fluorescence of 10 animals analyzed for each RNAi treatment; shown are the median and the first and third quartile. ‘ns’ indicates values not significantly higher than in unc-22(RNAi) animals (P ≥ 0.05; ANOVA). (E and F) Detection of SL1 trans-splicing inhibition by RT-qPCR. Animals carrying the Pvit-2::outron::GFPM1A transgene were subjected to sna-1, sna-2, sut-1 or unc-22(RNAi), and trans-splicing of gfp reporter gene and rps-3 transcripts was analyzed by reverse transcription followed by qPCR. The schematic diagrams indicate the position of the primers used (drawings are not to scale). Outron sequences are shown in blue and the SL1 acceptor sites in purple. Outron-gfp and outron-rps-3 RNA levels were standardized with respect to an internal part of the mRNA (internal) and levels in unc-22(RNAi) animals were defined as 1. Note that elevated outron-gfp or outron-rps-3 levels indicate inhibition of SL trans-splicing. ‘ns’ indicates values not significantly higher than in unc-22(RNAi) animals (P ≥ 0.05, t-test). Error bars show standard deviation from three technical replicates. Similar results were obtained in two independent experiments.
Figure 2.
Figure 2.
Loss-of-function mutations in sna-1 recovered from a mutagenesis screen for genes involved in SL1 trans-splicing. (A) Schematic representations of the location of the residues affected by the mutations in the SNA-1 protein. The regions in dark gray are conserved in nematode SNA-1 orthologues (Supplementary Figure S1). (B) Representative images of wild type (wt) and sna-1(fe47) animals transgenic for Pvit-2::outron::gfpM1A, showing intestinal GFP (green) expression and constitutive mCherry (red) expression in body wall muscle cells. Scale bar represents 100 μm. The graph plots the GFP fluorescence of 10 animals analyzed; shown are the median and the first and third quartile. (C and D) RT-qPCR analysis of SL1 trans-splicing in wt and sna-1(fe47) animals carrying the Pvit-2::outron::gfpM1A transgene. Trans-splicing of gfp reporter gene (C) and rps-3 transcripts (D) was analyzed as described in the legend of Figure 1, with levels in wt animals set as 1. Error bars indicate standard deviation from three technical replicates. (E) Viability of wt, sna-1(fe47) and sut-1(tm3079) animals at 20 and 15°C, as expressed by the proportion of eggs that develop to L4/adult stage in 5–10 independent experiments. (***P ≤ 0.001; ‘ns’ not significant; ANOVA). Note that while at 20°C there is a slight reduction in viability in sut-1(tm3079) animals, this is not significantly different from the viability of sna-1(fe47) animals.
Figure 3.
Figure 3.
SL1 spliced leader trans-splicing is sensitive to the knockdown of snr, but not lsm, gene transcripts. Detection of SL1 trans-splicing inhibition by fluorescence microscopy. Quantitation of GFP expression in animals carrying the Pvit-2::outron::GFPM1A transgene subjected to RNAi was done as described for Figure 1. (A) The proportion of GFP fluorescent animals. The number of animals analyzed are indicated above each dataset. (B) GFP fluorescence intensity. The graph summarizes individual measurements (n = 30) from three independent experiments. The distribution of measurements for each of the experiments is similar to the distribution of the pooled data. Median and the first and third quartile are indicated. ‘ns’ indicates values not significantly higher than in unc-22(RNAi) animals (P ≥ 0.05; ANOVA). (C and D) Detection of SL1 trans-splicing inhibition by RT-qPCR. Animals carrying the Pvit-2::outron::GFPM1A transgene were subjected to RNAi, and trans-splicing of the gfp reporter gene (C) and rps-3 transcripts (D) was analyzed by reverse transcription followed by qPCR. Outron-gfp and outron-rps-3 transcript levels were standardized as described in the Figure 1 legend, and levels in unc-22(RNAi) animals were defined as 1. Note that snr-7(RNAi) was not analyzed by RT-qPCR. ‘ns’ indicates values not significantly higher than in unc-22(RNAi) animals (P ≥ 0.05; t-test). Error bars show standard deviation from three technical replicates.
Figure 4.
Figure 4.
Knockdown of snRNP assembly factors inhibits SL1 spliced leader trans-splicing. Detection of SL1 trans-splicing inhibition by fluorescence microscopy. Quantitation of GFP expression in animals with the Pvit-2::outron::GFPM1A transgene subjected to RNAi was done as described for Figure 1. (A) The proportion of GFP fluorescent animals. The graph summarizes results from two or five independent experiments (unc-22(RNAi): 2; smn-1(RNAi), smi-1(RNAi), icln-1(RNAi): 5). The total number of animals analyzed is indicated. (B) GFP fluorescence intensity. The graph shows measurements of fluorescence intensity of 10 animals analyzed for each RNAi treatment. ns indicates values not significantly higher than in unc-22(RNAi) animals (P ≥ 0.05; ANOVA). (C and D) Detection of SL1 trans-splicing inhibition by RT-qPCR. Animals carrying the Pvit-2::outron::GFPM1A transgene were subjected to RNAi, and trans-splicing of the gfp reporter gene (C) and rps-3 transcripts (D) was analyzed by reverse transcription followed by qPCR. Outron-gfp and outron-rps-3 transcript levels were standardized as described in the Figure 1 legend, and levels in unc-22(RNAi) animals were defined as 1. ‘ns’ indicates values not significantly higher than in unc-22(RNAi) animals (P ≥ 0.05, t-test). Error bars show standard deviation from three technical replicates.
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References

    1. Pettitt J., Harrison N., Stansfield I., Connolly B., Müller B.. The evolution of spliced leader trans-splicing in nematodes. Biochem. Soc. Trans. 2010; 38:1125–1130. - PubMed
    1. Lasda E.L., Blumenthal T.. Trans-splicing. Wiley Interdiscip. Rev. RNA. 2011; 2:417–434. - PubMed
    1. Allen M.A., Hillier L.W., Waterston R.H., Blumenthal T.. A global analysis of C. elegans trans-splicing. Genome Res. 2011; 21:255–264. - PMC - PubMed
    1. Blumenthal T. Trans-Splicing and Operons in C. elegans. WormBook. 2012; The C. elegans Research Community. - PubMed
    1. Blumenthal T., Evans D., Link C.D., Guffanti A., Lawson D., Thierry-Mieg J., Thierry-Mieg D., Chiu W.L., Duke K., Kiraly M. et al. . A global analysis of Caenorhabditis elegans operons. Nature. 2002; 417:851–854. - PubMed

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