Dataset Information

Dinoflagellate tandem array gene transcripts are highly conserved and not polycistronic.

ABSTRACT: Dinoflagellates are an important component of the marine biota, but a large genome with high-copy number (up to 5,000) tandem gene arrays has made genomic sequencing problematic. More importantly, little is known about the expression and conservation of these unusual gene arrays. We assembled de novo a gene catalog of 74,655 contigs for the dinoflagellate Lingulodinium polyedrum from RNA-Seq (Illumina) reads. The catalog contains 93% of a Lingulodinium EST dataset deposited in GenBank and 94% of the enzymes in 16 primary metabolic KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways, indicating it is a good representation of the transcriptome. Analysis of the catalog shows a marked underrepresentation of DNA-binding proteins and DNA-binding domains compared with other algae. Despite this, we found no evidence to support the proposal of polycistronic transcription, including a marked underrepresentation of sequences corresponding to the intergenic spacers of two tandem array genes. We also have used RNA-Seq to assess the degree of sequence conservation in tandem array genes and found their transcripts to be highly conserved. Interestingly, some of the sequences in the catalog have only bacterial homologs and are potential candidates for horizontal gene transfer. These presumably were transferred as single-copy genes, and because they are now all GC-rich, any derived from AT-rich contexts must have experienced extensive mutation. Our study not only has provided the most complete dinoflagellate gene catalog known to date, it has also exploited RNA-Seq to address fundamental issues in basic transcription mechanisms and sequence conservation in these algae.

SUBMITTER: Beauchemin M

PROVIDER: S-EPMC3465430 | biostudies-literature | 2012 Sep

REPOSITORIES: biostudies-literature

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Dinoflagellate tandem array gene transcripts are highly conserved and not polycistronic.

Beauchemin Mathieu M Roy Sougata S Roy Sougata S Daoust Philippe P Dagenais-Bellefeuille Steve S Bertomeu Thierry T Letourneau Louis L Lang B Franz BF Morse David D

Proceedings of the National Academy of Sciences of the United States of America 20120910 39

Dinoflagellates are an important component of the marine biota, but a large genome with high-copy number (up to 5,000) tandem gene arrays has made genomic sequencing problematic. More importantly, little is known about the expression and conservation of these unusual gene arrays. We assembled de novo a gene catalog of 74,655 contigs for the dinoflagellate Lingulodinium polyedrum from RNA-Seq (Illumina) reads. The catalog contains 93% of a Lingulodinium EST dataset deposited in GenBank and 94% of ...[more]

PMID: 23019363

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Project description:Eukaryotic polycistronic transcription units are rare and only a few examples are known, mostly being the outcome of serendipitous discovery. We claim that nonsense-mediated mRNA decay (NMD) immune structure is a common characteristic of polycistronic transcripts, and that this immunity is an emergent property derived from all functional CDSs. The human RefSeq transcriptome was computationally screened for transcripts capable of eliciting NMD, and which contain an additional ORF(s) potentially capable of rescuing the transcript from NMD. Transcripts were further analyzed implementing domain-based strategies in order to estimate the potential of the candidate ORF to encode a functional protein. Consequently, we predict the existence of forty nine novel polycistronic transcripts. Experimental verification was carried out utilizing two different types of analyses. First, five Gene Expression Omnibus (GEO) datasets from published NMD-inhibition studies were used, aiming to explore whether a given mRNA is indeed insensitive to NMD. All known bicistronic transcripts and eleven out of the twelve predicted genes that were analyzed, displayed NMD insensitivity using various NMD inhibitors. For three genes, a mixed expression pattern was observed presenting both NMD sensitivity and insensitivity in different cell types. Second, we used published global translation initiation sequencing data from HEK293 cells to verify the existence of translation initiation sites in our predicted polycistronic genes. In five of our genes, the predicted rescuing uORFs are indeed identified as translation initiation sites, and in two additional genes, one of two predicted rescuing uORF is verified. These results validate our computational analysis and reinforce the possibility that NMD-immune architecture is a parameter by which polycistronic genes can be identified. Moreover, we present evidence for NMD-mediated regulation controlling the production of one or more proteins encoded in the polycistronic transcript.

Project description:The gene cassettes found in class 1 integrons are generally promoterless units composed by an open reading frame (ORF), a short 5' untranslated region (UTR) and a 3' recombination site ( attC). Fused gene cassettes are generated by partial or total loss of the attC from the first cassette in an array, creating, in some cases, a fusion with the ORF from the next cassette. These structures are rare and little is known about their mechanisms of mobilization and expression. The aim of this study was to evaluate the dynamic of mobilization and transcription of the gcu14-bla GES-1 /aacA4 gene cassette array, which harbours a fused gene cassette represented by bla GES-1 /aacA4. The cassette array was analyzed by Northern blot and real-time reverse transcription-polymerase chain reaction (RT-PCR) in order to assess the transcription mechanism of bla GES-1 /aacA4 fused cassette. Also, inverse polymerase chain reactions (PCR) were performed to detect the free circular forms of gcu14, bla GES-1 and aacA4. The Northern blot and real time RT-PCR revealed a polycistronic transcription, in which the fused cassette bla GES-1 /aacA4 is transcribed as a unique gene, while gcu14 (with a canonical attC recombination site) has a monocistronic transcription. The gcu14 cassette, closer to the weak configuration of cassette promoter (PcW), had a higher transcription level than bla GES-1/ aacA4, indicating that the cassette position affects the transcript amounts. The presence of ORF-11 at attI1, immediately preceding gcu14, and of a Shine-Dalgarno sequence upstream bla GES-1/ aacA4 composes a scenario for the occurrence of array translation. Inverse PCR generated amplicons corresponding to gcu14, gcu14-aacA4 and gcu14-bla GES-1/ aacA4 free circular forms, but not to bla GES-1 and aacA4 alone, indicating that the GES-1 truncated attC is not substrate of integrase activity and that these genes are mobilized together as a unique cassette. This study was original in showing the transcription of fused cassettes and in correlating cassette position with transcription.

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Dinoflagellate tandem array gene transcripts are highly conserved and not polycistronic.

Publications

Dinoflagellate tandem array gene transcripts are highly conserved and not polycistronic.

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