Cuypers B, Domagalska MA, Meysman P, Muylder G, Vanaerschot M, Imamura H, Dumetz F, Verdonckt TW, Myler PJ, Ramasamy G, Laukens K, Dujardin JC.

Sci Rep. 2017 Jun 16;7(1):3725. doi:10.1038/s41598-017-03987-0

Multiplexed Spliced-Leader Sequencing: A high-throughput, selective method for RNA-seq in Trypanosomatids.

[[!pmidi 28623350 desc="1 µg total RNA reverse-transcribed with SuperScript III and 2 µM random primer (GTATAAGAGACAGNNNNNNN). The RNA strand degraded with 2U RNAse H for 20 mi at 37 °C. The DNA strand was purified with Agencourt AMPure XP beads. 0.6 mM of SL primer (TCAGTTTCTGTA) was annealed to 25 µL of DNA from the previous step in 1x NEB buffer at 98 °C for 5 minutes and cooled down to room temperature for min. Second-strand synthesis with 5U Klenow fragment and 0.4 mM dNTPs in 50 µL at 37 °C for 60 minutes."]]

Derelle R, Momose T, Manuel M, Da Silva C, Wincker P, Houliston E.

RNA. 2010 Apr;16(4):696-707. doi:10.1261/rna.1975210

Convergent origins and rapid evolution of spliced leader trans-splicing in metazoa: insights from the ctenophora and hydrozoa.

“First evidence for SL trans-splicing in ctenophores.” (Pleurobrachia pileus and Mnemiopsis leidyi) “Analysis of data sets for the hydrozoans H. magnipapillata and C. hemisphaerica revealed a high number of SL sequence variants per species.” The same gene may use different splice leaders. An adenosine-rich region is found between the trans-splicing site and the transcription initiation site in hydrozoans, but not in other species (including Oikopleura.

Marius Wenzel, Berndt Mueller, Jonathan Pettitt

bioRxiv 2020.12.23.423594; doi:10.1101/2020.12.23.423594

SLIDR and SLOPPR: Flexible identification of spliced leader trans-splicing and prediction of eukaryotic operons from RNA-Seq data

Median intercistronic distance of 33 nt in Oikopleura. Calculated as the the distance between two “gene” annotations.

Bruzik JP, Maniatis T.

Nature. 1992 Dec 17;360(6405):692-5. doi:10.1038/360692a0

Spliced leader RNAs from lower eukaryotes are trans-spliced in mammalian cells.

Trans-splicing in vivo in COS cells and in vitro in HeLa cell extracts, using splice leaders and actin genes from C. elegans and Leptomonas colosoma, and also using an adenoviral RNA as acceptor.

Proc Natl Acad Sci U S A. 2016 Oct 25;113(43):12214-12219 doi:10.1073/pnas.1608016113

Nowack EC, Price DC, Bhattacharya D, Singer A, Melkonian M, Grossman AR.

Gene transfers from diverse bacteria compensate for reductive genome evolution in the chromatophore of Paulinella chromatophora.

Found splice leaders in P. chromatophora. Horizontal gene transfer as a possible mechanism facilitating gene loss in endosymbionts.

PLoS One. 2018 Jul 19;13(7):e0200961. doi:10.1371/journal.pone.0200961

Matsuo M, Katahata A, Satoh S, Matsuzaki M, Nomura M, Ishida KI, Inagaki Y, Obokata J.

Characterization of spliced leader trans-splicing in a photosynthetic rhizarian amoeba, Paulinella micropora, and its possible role in functional gene transfer.

Characterised two splice leaders in P. micropora, and discussed a possible link with endosymbiosis and horizontal gene transfer.

Matthysse AG, Deschet K, Williams M, Marry M, White AR, Smith WC.

A functional cellulose synthase from ascidian epidermis.

Proc Natl Acad Sci U S A. 2004 Jan 27;101(4):986-91.

The CesA mRNA is trans-spliced in Ciona savignyi. C. sav CesA can rescue mutant A. tumefaciens bacteria.

Vandenberghe AE, Meedel TH, Hastings KE.

mRNA 5'-leader trans-splicing in the chordates.

Genes Dev. 2001 Feb 1;15(3):294-303.

Discovery of trans-splicing in Tunicates.

Wang K, Omotezako T, Kishi K, Nishida H, Onuma TA.

Dev Genes Evol. 2015 Jun;225(3):149-59. doi:10.1007/s00427-015-0502-7

Maternal and zygotic transcriptomes in the appendicularian, Oikopleura dioica: novel protein-encoding genes, intra-species sequence variations, and trans-spliced RNA leader.

Only one splice leader was found and it was identical to the one reported by Ganot. “Samples were prepared from cohorts of a single pair.” “The transcript sequences showed a high degree of variability between the Japanese and Norwegian O. dioica populations. The average degrees of nucleotide and amino acid sequence conservation were 91.0 and 94.8 %, respectively.” “In the present analysis, the SL was observed in 40.8 % of mRNA species. It showed preferential linkage to adenine at the 5′ ends of the downstream exons. Intriguingly, the trans-splicing occurs more frequently in eggs than in larvae.” “Among the 12,311 assembled transcripts, 63 and 99 % were detected in eggs and larvae, respectively. Thus, the mRNAs of most of genes are present at the developing larval stage. In this quick developer, it is most probable that residual maternal mRNAs are still preserved in 8 hpf larvae.” “The raw data were deposited in the National Center for Biotechnology Information (NCBI) Short Read Archive (SRP accession number: SRP050571, run accession numbers are SRR1693762, SRR1693765, SRR1693766, and SRR1693767) and Gene Expression Omnibus (GEO accession: GSE64421).”

Yokomori R, Shimai K, Nishitsuji K, Suzuki Y, Kusakabe TG, Nakai K.

Genome Res. 2016 Jan;26(1):140-50. doi:10.1101/gr.184648.114

Genome-wide identification and characterization of transcription start sites and promoters in the tunicate Ciona intestinalis.

TSS-Seq libraries made of 200 µg of total RNA, sequenced on Illumina GA with a read length of 36, which is sufficient to go through the 16-nt splice leader.

Nucleic Acids Res. 2006 Jul 5;34(11):3378-88 doi:10.1093/nar/gkl418

Satou Y, Hamaguchi M, Takeuchi K, Hastings KE, Satoh N.

Genomic overview of mRNA 5'-leader trans-splicing in the ascidian Ciona intestinalis.

Analysis of oligo-capped cDNAs reports a single splice leader (16 nt-long) in 27% of the cDNAs.

Proc Natl Acad Sci U S A. 2004 Feb 10;101(6):1650-5. doi:10.1073/pnas.0308384100

Hwang BJ, Müller HM, Sternberg PW.

Genome annotation by high-throughput 5' RNA end determination.

TC-RED: primes with oligo-dT, amplifies primers complementary to the splice leader, cleaves tags with BpmI and sequences concatemers

Danks GB, Raasholm M, Campsteijn C, Long AM, Manak JR, Lenhard B, Thompson EM.

Mol Biol Evol. 2015 Mar;32(3):585-99. doi:10.1093/molbev/msu336

Trans-Splicing and Operons in Metazoans: Translational Control in Maternally Regulated Development and Recovery from Growth Arrest.

“39% of annotated gene models are trans-spliced with the SL.” “42% of SL transcripts are monocistronic; in these cases SL trans-splicing has a function other than resolving polycistronic mRNA.” “A second wave of gene activation peaked at the metamorphic tailshift stage where 2,578 genes were switched on for the first time. Here, 80% of genes were non-trans-spliced nonoperon genes, only 4% were SL-trans-spliced operon genes, and 10% were SL-trans-spliced monocistronic genes.” “A Switch from Predominantly Trans-Spliced to Predominantly Non-trans-Spliced Transcripts Occurs at the Maternal to Zygotic Transition” Parallel study in O. dioica, C. elegans and C. intestinalis suggests that “trans-splicing itself, rather than the organization of genes into operons, is the common factor for maternal mRNA in these metazoans”. The TOP motif of ribosomal protein genes is provided by the SL in O. dioica. “We find that SL-trans-spliced mRNAs are enriched in the ovary and oocytes.”

Ganot P, Kallesøe T, Reinhardt R, Chourrout D, Thompson EM.

Mol Cell Biol. 2004 Sep;24(17):7795-805. doi:10.1128/MCB.24.17.7795-7805.2004

Spliced-Leader RNA trans Splicing in a Chordate, Oikopleura dioica, with a Compact Genome.

5′ splice leader (SL) found in 90/158 ESTs containing a start codon. The SL RNA is found downstream of the 5S RNA in at least 40 occurences, and aproximately 2/3 of all the 5S rRNA genes. Its sequence is ACTCATCCCATTTTTGAGTCCGATTTCGATTGTCTAACAG. O. doica is the first chordate where gene operons have been described. Intercistronic regions are very short (<30 nt). “In comparing 52 distinct trans-splice acceptor sites to 605 cis-splice acceptor sites, the same consensus sequence, `TTT(C/T/A)AG`, was observed at both intron and intercistronic region 3′ ends. A notable difference from cis splicing was that most exons trans spliced to the leader (133 of 145) started with an adenosine, whereas the start of cis-spliced exons did not show any nucleotide preference.” In the cycD operon, only the last gene (cycD) has a `AAUAAA` polyadenylation signal.

Hybrid experiment found cases of trans-splicing, but few.