Molecular cloning and characterization of SL3: a stem cell-specific SL RNA from the planarian Schmidtea mediterranea

Abstract

Spliced leader (SL) trans-splicing is a biological phenomenon, common among many metazoan taxa, consisting in the transfer of a short leader sequence from a small SL RNA to the 5' end of a subset of pre-mRNAs. While knowledge of the biochemical mechanisms driving this process has accumulated over the years, the functional consequences of such post-transcriptional event at the organismal level remain unclear. In addition, the fact that functional analyses have been undertaken mainly in trypanosomes and nematodes leaves a somehow fragmented picture of the possible biological significance and evolution of SL trans-splicing in eukaryotes. Here, we analyzed the spatial expression of SL RNAs in the planarian flatworm Schmidtea mediterranea, with the goal of identifying novel developmental paradigms for the study of trans-splicing in metazoans. Besides the previously identified SL1 and SL2, S. mediterranea expresses a third SL RNA described here as SL3. While, SL1 and SL2 are collectively expressed in a broad range of planarian cell types, SL3 is highly enriched in a subset of the planarian stem cells engaged in regenerative responses. Our findings provide new opportunities to study how trans-splicing may regulate the phenotype of a cell.

Keywords: 0.15M NaCl/0.015M Na(3)·citrate pH7.6; 2,2,7-trimethylguanosine; 4′,6-diamidino-2-phenylindole; 5-bromo-4-chloro-3-indolyl phosphate; 7-methylguanosine; A; ATP; BAC; BCIP; C; CB; DAPI; DIG; DNA complementary to RNA; DNP; EST; FISH; FITC; G; IRR; Kb; MMG; NBT; Nitro Blue tetrazolium; OCT; Optimal Cutting Temperature compound; PBS; PCR; Planarians; R; RACE; RNP; RT-PCR; Regeneration; S; SDS; SL; SL RNA; SL RNP; SSC; Schmidtea mediterranea piwi-like protein; Sm; Smith's antigen; Stem cells; T; TMG; Trans-splicing; U; UTR; WT; adenosine; adenosine triphosphate; b; bacterial artificial chromosome; base pair(s); bp; brain; cDNA; cNeoblast; chromatoid bodies; clonogenic neoblast; cytidine; d; deoxyribo; digoxigenin; dinitrophenol; expressed sequence tag; fluorescein isothiocyanate; fluorescent in situ hybridization; g; guanosine; gut; in vitro transcribed standard; irradiation; kilobase(s) or 1000bp; mRNA; messenger RNA; nc; nerve chords; nt; nucleotide(s); p; pharynx; phosphate buffered saline; photoreceptors; polymerase chain reaction; pr; purine; rNTP; rRNA; rapid amplification of cDNA ends; reverse transcriptase-PCR; ribonucleoprotein; ribonucleotide; ribosomal RNA; sDMA; small nuclear RNA; smedwi-1; snRNA; sodium dodecyl sulfate; spliced leader; spliced leader RNA; spliced leader RNA ribonucleoprotein; symmetrical dimethylarginine; thymidine; untranslated region(s); uridine; wild type.