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Spliced leader RNA trans-splicing discovered in copepods.

Yang F, Xu D, Zhuang Y, Yi X, Huang Y, Chen H, Lin S, Campbell DA, Sturm NR, Liu G, Zhang H - Sci Rep (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: The Key Laboratory of Marine Environment and Ecology, Ministry of Education, Qingdao 266100, China.

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.

No MeSH data available.


Related in: MedlinePlus

(A) The gel image of the PCR products generated with primer set CopepodSL-Racer3. Left, lane M, DL5000 DNA Marker; (−), negative control; Acapa, Acartia pacifica, Calsi, Calanus sinicus, Labro, Labidocera rotunda; Cendo, Centropages dorsispinatus; Cente, Centropages tenuiremis; Parpa, Paracalanus parvus, Psepo, Pseudodiaptomus poplesia, Torde, Tortanus dextrilobatus and Torfo, Tortanus forcipatus. (B) Overall annotation result based on BLASTx hits (with E-value cutoff <10−5) of the nine copepod cDNAs against the NCBI nr database. No cDNAs from the potential organisms living on copepod exoskeleton were detected.
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f2: (A) The gel image of the PCR products generated with primer set CopepodSL-Racer3. Left, lane M, DL5000 DNA Marker; (−), negative control; Acapa, Acartia pacifica, Calsi, Calanus sinicus, Labro, Labidocera rotunda; Cendo, Centropages dorsispinatus; Cente, Centropages tenuiremis; Parpa, Paracalanus parvus, Psepo, Pseudodiaptomus poplesia, Torde, Tortanus dextrilobatus and Torfo, Tortanus forcipatus. (B) Overall annotation result based on BLASTx hits (with E-value cutoff <10−5) of the nine copepod cDNAs against the NCBI nr database. No cDNAs from the potential organisms living on copepod exoskeleton were detected.

Mentions: For a SL to be useful for transcriptomic studies, it has to exist in most, if not all, mRNAs of a species. To verify the ubiquity of CopepodSL in copepod mRNAs, we synthesized cDNAs for nine wild-caught copepod species representing seven families of the order Calanoida. PCR using CopepodSL-Racer3 primer set amplified cDNAs of 0.3–3 kb for all nine species, with 0.6–1 kb being the most abundant regardless of the difference of the original RNA quantity used in cDNA synthesis (Table 1, Fig. 2A). This indicated that the primer set CopepodSL-Racer3 was able to efficiently amplify cDNA libraries synthesized with very small amount of starting material (equivalent to 0.25 to 25 ng of total RNA). These cDNA amplicons were purified and cloned. We sequenced approximately 900 clones each for A. pacifica and P. poplesia, and 50–70 clones each for the other seven copepods. In total, 1,288 unique sequences were obtained (GenBank accession # KT754169 to KT755456). Rarefaction analysis31 indicated that the transcriptomic diversity has not been sampled exhaustively (See supplementary Fig. S2 online). Despite the limited sequencing depth, these cDNA datasets provided complete coding regions (cds) of the genes facilitating further gene characterization and functional annotation. These sequences were screened against the GenBank database, and 95% of them were identified as full-length cDNAs, while 5% had a complete 5′-end but missed the 3′-end likely due to the nonspecific binding of oligo-dT primer to a A-rich non-poly(A) tail region during cDNA synthesis. BLAST analysis of the cDNAs hit various protein genes mostly of copepods, insects and other animals; none of the cDNAs had top hits to genes of the organisms that were potential copepod prey (e.g., phytoplankton), or the symbionts living on or under the exoskeletons of copepods, such as ciliates and fungi16 (Fig. 2B, Supplementary Table S2 online). About 89% of A. pacifica and 84% of P. poplesia cDNAs were assigned putative gene function based on BLASTx search at E-value < 10−5. Gene ontology analysis showed diverse functions in each library (See Supplementary Fig. S3 online).


Spliced leader RNA trans-splicing discovered in copepods.

Yang F, Xu D, Zhuang Y, Yi X, Huang Y, Chen H, Lin S, Campbell DA, Sturm NR, Liu G, Zhang H - Sci Rep (2015)

(A) The gel image of the PCR products generated with primer set CopepodSL-Racer3. Left, lane M, DL5000 DNA Marker; (−), negative control; Acapa, Acartia pacifica, Calsi, Calanus sinicus, Labro, Labidocera rotunda; Cendo, Centropages dorsispinatus; Cente, Centropages tenuiremis; Parpa, Paracalanus parvus, Psepo, Pseudodiaptomus poplesia, Torde, Tortanus dextrilobatus and Torfo, Tortanus forcipatus. (B) Overall annotation result based on BLASTx hits (with E-value cutoff <10−5) of the nine copepod cDNAs against the NCBI nr database. No cDNAs from the potential organisms living on copepod exoskeleton were detected.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4664967&req=5

f2: (A) The gel image of the PCR products generated with primer set CopepodSL-Racer3. Left, lane M, DL5000 DNA Marker; (−), negative control; Acapa, Acartia pacifica, Calsi, Calanus sinicus, Labro, Labidocera rotunda; Cendo, Centropages dorsispinatus; Cente, Centropages tenuiremis; Parpa, Paracalanus parvus, Psepo, Pseudodiaptomus poplesia, Torde, Tortanus dextrilobatus and Torfo, Tortanus forcipatus. (B) Overall annotation result based on BLASTx hits (with E-value cutoff <10−5) of the nine copepod cDNAs against the NCBI nr database. No cDNAs from the potential organisms living on copepod exoskeleton were detected.
Mentions: For a SL to be useful for transcriptomic studies, it has to exist in most, if not all, mRNAs of a species. To verify the ubiquity of CopepodSL in copepod mRNAs, we synthesized cDNAs for nine wild-caught copepod species representing seven families of the order Calanoida. PCR using CopepodSL-Racer3 primer set amplified cDNAs of 0.3–3 kb for all nine species, with 0.6–1 kb being the most abundant regardless of the difference of the original RNA quantity used in cDNA synthesis (Table 1, Fig. 2A). This indicated that the primer set CopepodSL-Racer3 was able to efficiently amplify cDNA libraries synthesized with very small amount of starting material (equivalent to 0.25 to 25 ng of total RNA). These cDNA amplicons were purified and cloned. We sequenced approximately 900 clones each for A. pacifica and P. poplesia, and 50–70 clones each for the other seven copepods. In total, 1,288 unique sequences were obtained (GenBank accession # KT754169 to KT755456). Rarefaction analysis31 indicated that the transcriptomic diversity has not been sampled exhaustively (See supplementary Fig. S2 online). Despite the limited sequencing depth, these cDNA datasets provided complete coding regions (cds) of the genes facilitating further gene characterization and functional annotation. These sequences were screened against the GenBank database, and 95% of them were identified as full-length cDNAs, while 5% had a complete 5′-end but missed the 3′-end likely due to the nonspecific binding of oligo-dT primer to a A-rich non-poly(A) tail region during cDNA synthesis. BLAST analysis of the cDNAs hit various protein genes mostly of copepods, insects and other animals; none of the cDNAs had top hits to genes of the organisms that were potential copepod prey (e.g., phytoplankton), or the symbionts living on or under the exoskeletons of copepods, such as ciliates and fungi16 (Fig. 2B, Supplementary Table S2 online). About 89% of A. pacifica and 84% of P. poplesia cDNAs were assigned putative gene function based on BLASTx search at E-value < 10−5. Gene ontology analysis showed diverse functions in each library (See Supplementary Fig. S3 online).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: The Key Laboratory of Marine Environment and Ecology, Ministry of Education, Qingdao 266100, China.

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.

No MeSH data available.


Related in: MedlinePlus