23 research outputs found
Apparent non-canonical trans-splicing is generated by reverse transcriptase in vitro
Trans-splicing, the in vivo joining of two RNA molecules, is well characterized in several groups of simple organisms but was long thought absent from fungi, plants and mammals. However, recent bioinformatic analyses of expressed sequence tag (EST) databases suggested widespread trans-splicing in mammals^1-2^. Splicing, including the characterised trans-splicing systems, involves conserved sequences at the splice junctions. Our analysis of a yeast non-coding RNA revealed that around 30% of the products of reverse transcription lacked an internal region of 117 nt, suggesting that the RNA was spliced. The junction sequences lacked canonical splice-sites but were flanked by direct repeats, and further analyses indicated that the apparent splicing actually arose because reverse transcriptase can switch templates during transcription^3^. Many newly identified, apparently trans-spliced, RNAs lacked canonical splice sites but were flanked by short regions of homology, leading us to question their authenticity. Here we report that all reported categories of non-canonical splicing could be replicated using an in vitro reverse transcription system with highly purified RNA substrates. We observed the reproducible occurrence of ostensible trans-splicing, exon shuffling and sense-antisense fusions. The latter generate apparent antisense non-coding RNAs, which are also reported to be abundant in humans^4^. Different reverse transcriptases can generate different products of template switching, providing a simple diagnostic. Many reported examples of splicing in the absence of canonical splicing signals may be artefacts of cDNA preparation
Analysis of Expressed Sequence Tags of the Cyclically Parthenogenetic Rotifer Brachionus plicatilis
Background. Rotifers are among the most common non-arthropod animals and are the most experimentally tractable members of the basal assemblage of metazoan phyla known as Gnathifera. The monogonont rotifer Brachionus plicatilis is a developing model system for ecotoxicology, aquatic ecology, cryptic speciation, and the evolution of sex, and is an important food source for finfish aquaculture. However, basic knowledge of the genome and transcriptome of any rotifer species has been lacking. Methodology/Principal Findings. We generated and partially sequenced a cDNA library from B. plicatilis and constructed a database of over 2300 expressed sequence tags corresponding to more than 450 transcripts. About 20% of the transcripts had no significant similarity to database sequences by BLAST; most of these contained open reading frames of significant length but few had recognized Pfam motifs. Sixteen transcripts accounted for 25% of the ESTs; four of these had no significant similarity to BLAST or Pfam databases. Putative up- and downstream untranslated regions are relatively short and AT rich. In contrast to bdelloid rotifers, there was no evidence of a conserved trans-spliced leader sequence among the transcripts and most genes were single-copy. Conclusions/Significance. Despite the small size of this EST project it revealed several important features of the rotifer transcriptome and of individual monogonont genes. Because there is little genomic data for Gnathifera, the transcripts we found with no known function may represent genes that are species-, class-, phylum- or even superphylum-specific; the fact that some are among the most highly expressed indicates their importance. The absence of trans-spliced leader exons in this monogonont species contrasts with their abundance in bdelloid rotifers and indicates that the presence of this phenomenon can vary at the subphylum level. Our EST database provides a relatively large quantity of transcript-level data for B. plicatilis, and more generally of rotifers and other gnathiferan phyla, and can be browsed and searched at gmod.mbl.edu
Discovery of Nuclear-Encoded Genes for the Neurotoxin Saxitoxin in Dinoflagellates
Saxitoxin is a potent neurotoxin that occurs in aquatic environments worldwide.
Ingestion of vector species can lead to paralytic shellfish poisoning, a severe
human illness that may lead to paralysis and death. In freshwaters, the toxin is
produced by prokaryotic cyanobacteria; in marine waters, it is associated with
eukaryotic dinoflagellates. However, several studies suggest that saxitoxin is
not produced by dinoflagellates themselves, but by co-cultured bacteria. Here,
we show that genes required for saxitoxin synthesis are encoded in the nuclear
genomes of dinoflagellates. We sequenced >1.2×106 mRNA
transcripts from the two saxitoxin-producing dinoflagellate strains
Alexandrium fundyense CCMP1719 and A.
minutum CCMP113 using high-throughput sequencing technology. In
addition, we used in silico transcriptome analyses, RACE, qPCR
and conventional PCR coupled with Sanger sequencing. These approaches
successfully identified genes required for saxitoxin-synthesis in the two
transcriptomes. We focused on sxtA, the unique starting gene of
saxitoxin synthesis, and show that the dinoflagellate transcripts of
sxtA have the same domain structure as the cyanobacterial
sxtA genes. But, in contrast to the bacterial homologs, the
dinoflagellate transcripts are monocistronic, have a higher GC content, occur in
multiple copies, contain typical dinoflagellate spliced-leader sequences and
eukaryotic polyA-tails. Further, we investigated 28 saxitoxin-producing and
non-producing dinoflagellate strains from six different genera for the presence
of genomic sxtA homologs. Our results show very good agreement
between the presence of sxtA and saxitoxin-synthesis, except in
three strains of A. tamarense, for which we amplified
sxtA, but did not detect the toxin. Our work opens for
possibilities to develop molecular tools to detect saxitoxin-producing
dinoflagellates in the environment
REGIONAL DIFFERENCES IN TROPONIN-I ISOFORM SWITCHING DURING RAT-HEART DEVELOPMENT
Expression of cardiac troponin I (TnIcardiac) and slow skeletal troponin I
(TnIslow) genes was analyzed at the mRNA and protein level in the developing rat
heart. TnIslow mRNA was detectable by in situ hybridization in the embryonic
cardiac tube as early as the 13-somite stage (Embryonic Day 10). In contrast,
TnIcardiac transcripts were first detected in the primordial atrium and ventricle
of 11-day-old embryos, but were absent in the outflow tract region. TnIslow mRNA
levels decreased after birth in atria and later in ventricles but persisted even
in adult life in myocytes of the conduction system. TnIslow protein was detected
by specific antibodies in atrial myocytes beginning from Embryonic Day 11; in
contrast, ventricular myocytes were unreactive until Embryonic Day 18. Western
blot analysis of 16-day-old fetal hearts confirmed the expression of TnIcardiac
in atrial but not in ventricular myocardium. Slot blot analysis showed that at
this stage equivalent amounts of TnIslow and TnIcardiac mRNAs are expressed in
atria and ventricles. Similar differences in the expression of TnIslow and
TnIcardiac mRNAs and proteins were observed in cultures of embryonic atrial and
ventricular myocytes. The results suggest serial rather than simultaneous
activation of TnIslow and TnIcardiac genes and they show that different regions
of the developing heart differ in their patterns of TnIcardiac expression due to
the operation of distinct mechanisms that separately affect the accumulation of
TnIcardiac mRNA and protein