Abstract
Copepods are one of the most abundant metazoans in the marine ecosystem, constituting a critical link in aquatic food webs and contributing significantly to the global carbon budget, yet molecular mechanisms of their gene expression are not well understood. Here we report the detection of spliced leader (SL) trans-splicing in calanoid copepods. We have examined nine species of wild-caught copepods from Jiaozhou Bay, China that represent the major families of the calanoids. All these species contained a common 46-nt SL (CopepodSL). We further determined the size of CopepodSL precursor RNA (slRNA; 108-158 nt) through genomic analysis and 3′-RACE technique, which was confirmed by RNA blot analysis. Structure modeling showed that the copepod slRNA folded into typical slRNA secondary structures. Using a CopepodSL-based primer set, we selectively enriched and sequenced copepod full-length cDNAs, which led to the characterization of copepod transcripts and the cataloging of the complete set of 79 eukaryotic cytoplasmic ribosomal proteins (cRPs) for a single copepod species. We uncovered the SL trans-splicing in copepod natural populations, and demonstrated that CopepodSL was a sensitive and specific tool for copepod transcriptomic studies at both the individual and population levels and that it would be useful for metatranscriptomic analysis of copepods.
Highlights
Symbiotic or parasitic organisms on or under the chitinous skeleton and appendages of copepods, such as ciliates, fungi, diatoms, dinoflagellates[16], as well as prey organisms in the gut
Thirty-three of these different transcripts, encoding various proteins such as ribosomal proteins, 14-3-3, carbonic anhydrase 2, and proteasome subunit beta type 7 precursor, had a nearly identical 46-nt sequence at the beginning of the 5′ -untranslated region (UTR), indicating that this 46-nt sequence is a spliced leader trans-spliced to the 5′ -end of mRNAs in A. pacifica (Fig. 1A)
CopepodSL is a bona fide addition because 1) the conserved sequences occur at the 5′ end of mRNAs of diverse genes in calanoid copepods, 2) the gene encoding CopepodSL was isolated, and RNA blot analysis confirmed the existence of corresponding RNA, and 3) structural simulation showed typical folding of slRNA
Summary
Symbiotic or parasitic organisms on or under the chitinous skeleton and appendages of copepods, such as ciliates, fungi, diatoms, dinoflagellates[16], as well as prey organisms in the gut. Full-length cDNA sequences have proven to be informative[24,25,26,27], as complete coding regions allow for more accurate functional annotations and gene identification in the genome This is especially true for gene function prediction of non-model species or field-collected samples when a large portion of the transcriptome sequences do not have clear hits to the reported sequences in DNA and protein databases[18,27]. Proof of the existence of SL trans-splicing in a given lineage of organisms is not readily available from the existing transcriptomic data reported in the databases Because most of these data were not obtained using methods targeting the full-length sequence of the transcripts, the sequence information of the very 5′ -end where the SL resides is missing or truncated in almost all cases (see Zhang et al.[27] and the references therein). Through SL-based cDNA library construction and sequencing, we showed that this copepod SL could be a sensitive and specific tool in calanoid copepod transcriptomic studies
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