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. 2014 Jan 1;533(1):156-67.
doi: 10.1016/j.gene.2013.09.101. Epub 2013 Oct 8.

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

Alessandro Rossi  1 , Eric J Ross, Antonia Jack, Alejandro Sánchez Alvarado
Affiliations

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

Alessandro Rossi et al. Gene. .

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.

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Figures

Fig. 1
Fig. 1
Identification of the novel SL RNA SL3 in S. mediterranea. (A) Schematic representation of the SL trans-splicing reaction. The SL RNA is a small RNA containing a splicing donor site (GU) downstream of the spliced leader (in red). The donor site is engaged in a splicing reaction with the acceptor sites (AG) of target pre-mRNAs. In most of the metazoan examined to date the SL RNA is capped with a 2,2,7-trimethylguanosine (TMG) group. Consequently, mRNAs appended to the SL are also TMG-capped (Lasda and Blumenthal, 2011). Conversely, non trans-spliced transcripts possess a mono-methylated cap (MMG). In planarians, mRNAs competent for trans-splicing can also acquire a 5′ UTR in cis via splicing to an upstream exon or retention of an outron. (B) ClustalW alignment of the three known planarian SL RNAs. (C) MFOLD prediction of SL3 secondary structure. (D) Northern blot analysis of SL RNAs and trans-spliced mRNAs. The SL RNAs are indicated by the black arrow, while the rest of the signal corresponds to trans-spliced transcripts. (B–C) The spliced leader–intron boundary is indicated by the red arrowhead and the Sm protein binding site (consensus RAU4–6GR) (Scofield and Lynch, 2008) is underlined in red.
Fig. 2
Fig. 2
SL1 and SL3 are tissue-specifically expressed in S. mediterranea. Whole mount in situ hybridization of wild type worms hybridized with SL1/2 (A–C) and SL3 (G–I) riboprobes. In situ processed worms were also sectioned (D–F, J–L). Different areas of the same specimen and representative transverse sections across the same region are shown as indicated: A, D, G, and J cephalic region. B, E, H, and K trunk region. C, F, I, and L caudal region. Indicated in red characters are: brain (b); nerve chords (nc); pharynx (p); gut (g); photoreceptors (pr). Scale bars, 50 μm.
Fig. 3
Fig. 3
SL3 expression is affected by irradiation. (A) Wild type worms (WT) were subjected to 10Krads of γ-irradiation (IRR) and processed for in situ hybridization and Northern blot 24 h later. Irradiation completely depletes neoblasts under these conditions (smedwi-1, IRR). RNA levels for SL1/2 and SL3 were quantified by Northern blot (bottom of the situ hybridization panels). Scale bars, 100 μm.
Fig. 4
Fig. 4
FISH analysis confirms the overall broad expression of SL1 and the transcription of SL1 and SL3 in the neoblasts. Wild type specimens were processed for double FISH with either SL1/smedwi-1 (A–K) or SL3/smedwi-1 (L–M, O–R) riboprobe combinations. In N and S–V panels SL3 FISH was followed by immunostaining with the Y12 antibody. At the end of the staining procedures nuclei were counterstained with DAPI (D, H, O, S). (A–C) Low magnification images of a specimen hybridized with SL1 (red) and smedwi-1 (green) riboprobes (C, merge). (D–G) Higher magnification images of SL1 expression in the brain lobes (b) and in the mesenchyme between the brain (indicated by *). The SL1 riboprobe (red) decorates the nuclei (blue) of most cells including the neoblasts (green) (G, merge). (H–K) Most of the neoblasts (green) in the mesenchyme express SL1 (red) (K, merge). (L–M) A specimen double labeled with SL3 (red) and smedwi-1+ (green) imaged at low magnification. (N) The Y12 antibody (green) preferentially reacts with the chromatoid bodies (CBs) in the neoblasts, yielding a pattern similar to smedwi-1 in situ hybridizations. Shown is a worm stained after SL3 FISH (signal not shown at this magnification). (O–R) SL3 (red) is expressed in a subset of smedwi-1+ cells (green) (R, merge). Indicated in O are the nuclei of smedwi-1−/SL3+ (1), smedwi-1+/SL3+ (2) and smedwi-1+/SL3− (3) cells. (S–V) SL3 RNA (red) does not colocalize with CBs (green) (V, merge). Images were taken with 5× (A–C and L–N), 20× (D–K and O–R) or40× (S–V) objectives. Scale bars: A–C and L–N, 165μm; D–G, 25μm; H–K and O–V, 10μm.
Fig. 5
Fig. 5
Regeneration induces accumulation of SL3+ cells at wound sites and an increase in whole animal SL3 RNA levels. (A) Wild type worms were amputated in pre- and post-pharyngeal regions. Resulting trunks were allowed to regenerate and processed for in situ hybridization at the indicated time points. Regenerating photoreceptors at day 7 are circled. Scale bars, 50 μm. (B) Northern blot analysis of RNA obtained from uncut worms and regenerating trunks. Fold changes relative to uncut animals and normalized to the U6 loading control are indicated for SL1/2 and SL3 blots. (C) Example of coronal sections used to quantify the number of smedwi-1+ (green) and SL3+ (red) in the post blastema (Table S3). Nuclei (blue) are stained with DAPI and brain lobes (b) are indicated. Scale bars, 25 μm.
Fig. 6
Fig. 6
Expression of SL3 during colony expansion. Shown are representative colonies of increasing size and the percentage of smedwi-1+ cells (green) expressing SL3 (red). The size of 24 colonies from worms fixed 5–12 days after non-lethal irradiation and their content in SL3+ neoblasts were plotted. The percentage of smedwi-1+ cells expressing SL3 in wild type animals is indicated by the dotted line.
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