Computational analysis of spliced leader trans-splicing in the regenerative flatworm <i>Macrostomum lignano</i> reveals its prevalence in conserved and stem cell related genes

Many trypanosome genes are expressed as part of large polycistronic transcription units. This finding suggests that regulation of mRNA biogenesis may emphasize RNA-processing reactions more so than in other organisms. This study was undertaken to understand the temporal order of two RNA-processing reactions, trans splicing and polyadenylation, in the maturation of trypanosome mRNAs in vivo. Kinetic studies revealed rapid trans splicing of alpha-tubulin, beta-tubulin, and actin pre-mRNAs within 1 to 2 min after synthesis of the 3' splice site. Furthermore, following blockage of pre-mRNA synthesis, newly synthesized spliced leader RNA cannot be used for trans splicing, suggesting that trypanosomes do not accumulate substantial amounts of pre-mRNA which can provide splice acceptor sites. Thus, trans splicing is cotranscriptional. In addition, we show that trans splicing precedes polyadenylation in the processing of trypanosome tubulin pre-mRNAs.

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Minimum free energy predicted base pairing in the 39 nt spliced leader and 5’ UTR of calmodulin mRNA from Trypanosoma cruzi: influence of the multiple trans-splicing sites

Anais da Academia Brasileira de Ciências ◽

10.1590/0001-3765201720170082 ◽

2018 ◽

Vol 90 (2 suppl 1) ◽

pp. 2311-2316

Author(s):

FRANKLYN SAMUDIO ◽

ADEILTON BRANDÃO

Keyword(s):

Free Energy ◽

Trypanosoma Cruzi ◽

Minimum Free Energy ◽

Base Pairing ◽

Spliced Leader ◽

Trans Splicing

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Evaluation of various RNA-seq approaches for identification of gene outrons in the flatworm Opisthorchis felineus

Vavilov Journal of Genetics and Breeding ◽

10.18699/vj20.688 ◽

2020 ◽

Vol 24 (8) ◽

pp. 897-904

Author(s):

N. I. Ershov ◽

D. E. Maslov ◽

N. P. Bondar

Keyword(s):

High Efficiency ◽

The Other ◽

Opisthorchis Felineus ◽

Transcription Start Sites ◽

Spliced Leader ◽

Trans Splicing ◽

Rrna Depletion ◽

Causative Agents ◽

Bona Fide ◽

Nascent Transcripts

The parasitic flatworm Opisthorchis felineus is one of the causative agents of opisthorchiasis in humans. Recently, we assembled the O. felineus genome, but the correct genome annotation by means of standard methods was hampered by the presence of spliced leader trans-splicing (SLTS). As a result of SLTS, the original 5’-end (outron) of the transcripts is replaced by a short spliced leader sequence donated from a specialized SL RNA. SLTS is involved in the RNA processing of more than half of O. felineus genes, making it hard to determine the structure of outrons and bona fide transcription start sites of the corresponding genes and operons, being based solely on mRNA-seq data. In the current study, we tested various experimental approaches for identifying the sequences of outrons in O. felineus using massive parallel sequencing. Two of them were developed by us for targeted sequencing of already processed branched outrons. One was based on sequence-specific reverse transcription from the SL intron toward the 5’-end of the Y-branched outron. The other used outron hybridization with an immobilized single-stranded DNA probe complementary to the SL intron. Additionally, two approaches to the sequencing of rRNA-depleted total RNA were used, allowing the identification of a wider range of transcripts compared to mRNAseq. One is based on the enzymatic elimination of overrepresented cDNAs, the other utilizes exonucleolytic degradation of uncapped RNA by Terminator enzyme. By using the outron-targeting methods, we were not able to obtain the enrichment of RNA preparations by processed outrons, which is most likely indicative of a rapid turnover of these trans-splicing intermediate products. Of the two rRNA depletion methods, a method based on the enzymatic normalization of cDNA (Zymo-Seq RiboFree) showed high efficiency. Compared to mRNA-seq, it provides an approximately twofold increase in the fraction of reads originating from outrons and introns. The results suggest that unprocessed nascent transcripts are the main source of outron sequences in the RNA pool of O. felineus.

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Trans-splicing of mRNAs links gene transcription to translational control regulated by mTOR

10.1101/353979 ◽

2018 ◽

Author(s):

Gemma Danks ◽

Heloisa Galbiati ◽

Martina Raasholm ◽

Yamila N. Torres Cleuren ◽

Eivind Valen ◽

...

Keyword(s):

Gene Transcription ◽

Growth Regulator ◽

Translational Control ◽

Oikopleura Dioica ◽

Spliced Leader ◽

Animal Density ◽

Genome Wide ◽

Trans Splicing ◽

Initial Recovery ◽

Favorable Conditions

AbstractIn phylogenetically diverse organisms, the 5’ ends of a subset of mRNAs are trans-spliced with a spliced leader (SL) RNA. The functions of SL trans-splicing, however, remain largely enigmatic. Here, we quantified translation genome-wide in the marine chordate, Oikopleura dioica, under inhibition of mTOR, a central growth regulator. Translation of trans-spliced TOP mRNAs was suppressed, showing that the SL sequence permits nutrient-dependent translational control of growth-related mRNAs. Under crowded, nutrient-limiting conditions, O. dioica continues to filter-feed, but arrests growth until favorable conditions return. Upon release from such conditions, initial recovery was independent of nutrient-responsive, trans-spliced genes, suggesting animal density sensing as a first trigger for resumption of development. Our results demonstrate a role for trans-splicing in the coordinated translational down-regulation of nutrient-responsive genes under limiting conditions.

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