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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) Example of CopepodSL identified in various cDNAs recovered from Acartia pacifica GeneRacer library (Acapa) and a previously reported Calanus glacialis heat shock protein 60 cDNA sequence (*Calgl HSP60). (B,C) The genomic structures of the major types of the CopepodSL encoding genes in A. pacifica (B) and P. poplesia (C). (D,E,F) Image of total RNAs from A. pacifica (lane 1), Calanus sinicus (lane 2) and Pseudodiaptomus poplesia (lane 3) electrophoresed in an 8% acrylamide and 8M urea gel (D), and the Northern Blots hybridized with 32P-labeled CopepodSLa/s (E,F). Arrows indicate the apparent single thick bands under milder washing condition (E; 2 × SSC at 43ºC) were composed of two bands with slightly different sizes as seen under stringent washing (F; 2 × SSC at 50ºC). Total RNA from Leishmania tarentolae cells was co-electrophoresed (not shown) to provide known size markers18.
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f1: (A) Example of CopepodSL identified in various cDNAs recovered from Acartia pacifica GeneRacer library (Acapa) and a previously reported Calanus glacialis heat shock protein 60 cDNA sequence (*Calgl HSP60). (B,C) The genomic structures of the major types of the CopepodSL encoding genes in A. pacifica (B) and P. poplesia (C). (D,E,F) Image of total RNAs from A. pacifica (lane 1), Calanus sinicus (lane 2) and Pseudodiaptomus poplesia (lane 3) electrophoresed in an 8% acrylamide and 8M urea gel (D), and the Northern Blots hybridized with 32P-labeled CopepodSLa/s (E,F). Arrows indicate the apparent single thick bands under milder washing condition (E; 2 × SSC at 43ºC) were composed of two bands with slightly different sizes as seen under stringent washing (F; 2 × SSC at 50ºC). Total RNA from Leishmania tarentolae cells was co-electrophoresed (not shown) to provide known size markers18.

Mentions: Approximately 2 μg of Acartia pacifica mRNA was used to construct a library with enriched full-length cDNAs using a GeneRacerTM Kit (Invitrogen, USA). Out of 327 high-quality sequences, 124 sequences contained a complete coding region, totally representing 61 unique genes7. 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 (named CopepodSL hereafter, Table 1) is a spliced leader trans-spliced to the 5′-end of mRNAs in A. pacifica (Fig. 1A). Three of the full-length cDNAs had an additional three nucleotides, ATT, at the 5′-end of the CopepodSL.


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) Example of CopepodSL identified in various cDNAs recovered from Acartia pacifica GeneRacer library (Acapa) and a previously reported Calanus glacialis heat shock protein 60 cDNA sequence (*Calgl HSP60). (B,C) The genomic structures of the major types of the CopepodSL encoding genes in A. pacifica (B) and P. poplesia (C). (D,E,F) Image of total RNAs from A. pacifica (lane 1), Calanus sinicus (lane 2) and Pseudodiaptomus poplesia (lane 3) electrophoresed in an 8% acrylamide and 8M urea gel (D), and the Northern Blots hybridized with 32P-labeled CopepodSLa/s (E,F). Arrows indicate the apparent single thick bands under milder washing condition (E; 2 × SSC at 43ºC) were composed of two bands with slightly different sizes as seen under stringent washing (F; 2 × SSC at 50ºC). Total RNA from Leishmania tarentolae cells was co-electrophoresed (not shown) to provide known size markers18.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: (A) Example of CopepodSL identified in various cDNAs recovered from Acartia pacifica GeneRacer library (Acapa) and a previously reported Calanus glacialis heat shock protein 60 cDNA sequence (*Calgl HSP60). (B,C) The genomic structures of the major types of the CopepodSL encoding genes in A. pacifica (B) and P. poplesia (C). (D,E,F) Image of total RNAs from A. pacifica (lane 1), Calanus sinicus (lane 2) and Pseudodiaptomus poplesia (lane 3) electrophoresed in an 8% acrylamide and 8M urea gel (D), and the Northern Blots hybridized with 32P-labeled CopepodSLa/s (E,F). Arrows indicate the apparent single thick bands under milder washing condition (E; 2 × SSC at 43ºC) were composed of two bands with slightly different sizes as seen under stringent washing (F; 2 × SSC at 50ºC). Total RNA from Leishmania tarentolae cells was co-electrophoresed (not shown) to provide known size markers18.
Mentions: Approximately 2 μg of Acartia pacifica mRNA was used to construct a library with enriched full-length cDNAs using a GeneRacerTM Kit (Invitrogen, USA). Out of 327 high-quality sequences, 124 sequences contained a complete coding region, totally representing 61 unique genes7. 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 (named CopepodSL hereafter, Table 1) is a spliced leader trans-spliced to the 5′-end of mRNAs in A. pacifica (Fig. 1A). Three of the full-length cDNAs had an additional three nucleotides, ATT, at the 5′-end of the CopepodSL.

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