Limits...
A large family of Dscam genes with tandemly arrayed 5' cassettes in Chelicerata.

Yue Y, Meng Y, Ma H, Hou S, Cao G, Hong W, Shi Y, Guo P, Liu B, Shi F, Yang Y, Jin Y - Nat Commun (2016)

Bottom Line: Furthermore, extraordinary isoform diversity has been generated through a combination of alternating promoter and alternative splicing.These sDscams have a high sequence similarity with Drosophila Dscam1, and share striking organizational resemblance to the 5' variable regions of vertebrate clustered Pcdhs.Hence, our findings have important implications for understanding the functional similarities between Drosophila Dscam1 and vertebrate Pcdhs, and may provide further mechanistic insights into the regulation of isoform diversity.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biochemistry, Innovation Center for Signaling Network, College of Life Sciences, Zhejiang University, Hangzhou, Zhejiang ZJ310058, China.

ABSTRACT
Drosophila Dscam1 (Down Syndrome Cell Adhesion Molecules) and vertebrate clustered protocadherins (Pcdhs) are two classic examples of the extraordinary isoform diversity from a single genomic locus. Dscam1 encodes 38,016 distinct isoforms via mutually exclusive splicing in D. melanogaster, while the vertebrate clustered Pcdhs utilize alternative promoters to generate isoform diversity. Here we reveal a shortened Dscam gene family with tandemly arrayed 5' cassettes in Chelicerata. These cassette repeats generally comprise two or four exons, corresponding to variable Immunoglobulin 7 (Ig7) or Ig7-8 domains of Drosophila Dscam1. Furthermore, extraordinary isoform diversity has been generated through a combination of alternating promoter and alternative splicing. These sDscams have a high sequence similarity with Drosophila Dscam1, and share striking organizational resemblance to the 5' variable regions of vertebrate clustered Pcdhs. Hence, our findings have important implications for understanding the functional similarities between Drosophila Dscam1 and vertebrate Pcdhs, and may provide further mechanistic insights into the regulation of isoform diversity.

No MeSH data available.


Related in: MedlinePlus

Highly complex combinations of sDscam 5′ variable exons.(a) Schematic diagram for splicing patterns of the 5′ variable exons. Symbols used are the same as in Fig. 1. Canonical splicing isoforms were joined in neighbouring junctions in variable cassettes, according to the previous ‘cap-proximal splicing' model19. Non-canonical splicing isoforms included: (I) splicing isoforms from the same cassette with either exon 2, 3 or 4 skipped; (II) splicing isoforms that contained variable exons from tandem cassettes; as well as (III) the isoforms that contained within-cassette introns. (b) Quantification of the canonical and non-canonical splicing isoforms. (c) Schematic diagram of the splicing patterns of the 5′ variable exons in M. martensii sDscamβ1. Splice isoforms within a single tandem cassette are shown as a black line above the gene structure diagram, while splice isoforms from different tandem cassettes are represented below by coloured lines. (d) Alternative splicing junctions from different cassettes were validated using reverse transcription–PCR (RT–PCR). Due to the low expression of sDscam variable exons, nested PCR was necessary to amplify the products; only the primers used in the second PCR are depicted and same in panels below. The RT–PCR products were confirmed by cloning and sequencing. These experiments revealed the splicing of multiple cassette variants from different tandem cassettes. (e) Splicing patterns of the 5′ variable exons in sDscamβ3. (f) RT–PCR was used to detect alternative splice isoforms in sDscamβ3. (g) A summary of several types of isoforms with distinct Ig numbers generated by alternative splicing.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4835542&req=5

f5: Highly complex combinations of sDscam 5′ variable exons.(a) Schematic diagram for splicing patterns of the 5′ variable exons. Symbols used are the same as in Fig. 1. Canonical splicing isoforms were joined in neighbouring junctions in variable cassettes, according to the previous ‘cap-proximal splicing' model19. Non-canonical splicing isoforms included: (I) splicing isoforms from the same cassette with either exon 2, 3 or 4 skipped; (II) splicing isoforms that contained variable exons from tandem cassettes; as well as (III) the isoforms that contained within-cassette introns. (b) Quantification of the canonical and non-canonical splicing isoforms. (c) Schematic diagram of the splicing patterns of the 5′ variable exons in M. martensii sDscamβ1. Splice isoforms within a single tandem cassette are shown as a black line above the gene structure diagram, while splice isoforms from different tandem cassettes are represented below by coloured lines. (d) Alternative splicing junctions from different cassettes were validated using reverse transcription–PCR (RT–PCR). Due to the low expression of sDscam variable exons, nested PCR was necessary to amplify the products; only the primers used in the second PCR are depicted and same in panels below. The RT–PCR products were confirmed by cloning and sequencing. These experiments revealed the splicing of multiple cassette variants from different tandem cassettes. (e) Splicing patterns of the 5′ variable exons in sDscamβ3. (f) RT–PCR was used to detect alternative splice isoforms in sDscamβ3. (g) A summary of several types of isoforms with distinct Ig numbers generated by alternative splicing.

Mentions: Inconsistent with the presence of a large first exon in the clustered Pcdh gene4, a cassette repeat composed of two or four exons was identified in the clustered sDscam gene. This raised the question of how these variable exons were combined into distinct mRNA isoforms, particularly because the exclusion or multiple inclusions of exons 2, 3 or 4 variants would not result in a frameshift. To explore this, we defined exon junctions based on a total of 0.7 billion RNA-seq reads from different tissues. At least 264 distinct exon junctions were detected, 249 of which were joined neighbouring junctions in single tandem cassettes. This suggests that most isoforms could be made through joining neighbouring junctions in variable cassette regions. Moreover, we detected a small fraction of isoforms from the same cassette with either exon 2, 3 and/or 4 skipped. In these cases, the variable exon skipping resulted in an incomplete Ig domain (that is, the sDscamβ6 variable exon 3.1) (Fig. 5a). This abnormal splicing is analogous to the skipping of Dscam exon 4 variants, which results in a partial Ig2 domain and is likely to be biologically relevant28. In addition, we detected other non-canonical splicing isoforms that contained variable exons from different tandem cassettes, as well as the isoforms containing within-cassette introns (Fig. 5a). Based on the exon junctions from the RNA-seq data, we estimated that ∼10–40% of isoforms resulted from non-canonical splicing in most sDscam genes, which showed differential expression in various tissues (Fig. 5a,b; Supplementary Fig. 10a,b). Taken together, these data indicate that sDscams have potentially complex splicing patterns at the 5′ variable regions.


A large family of Dscam genes with tandemly arrayed 5' cassettes in Chelicerata.

Yue Y, Meng Y, Ma H, Hou S, Cao G, Hong W, Shi Y, Guo P, Liu B, Shi F, Yang Y, Jin Y - Nat Commun (2016)

Highly complex combinations of sDscam 5′ variable exons.(a) Schematic diagram for splicing patterns of the 5′ variable exons. Symbols used are the same as in Fig. 1. Canonical splicing isoforms were joined in neighbouring junctions in variable cassettes, according to the previous ‘cap-proximal splicing' model19. Non-canonical splicing isoforms included: (I) splicing isoforms from the same cassette with either exon 2, 3 or 4 skipped; (II) splicing isoforms that contained variable exons from tandem cassettes; as well as (III) the isoforms that contained within-cassette introns. (b) Quantification of the canonical and non-canonical splicing isoforms. (c) Schematic diagram of the splicing patterns of the 5′ variable exons in M. martensii sDscamβ1. Splice isoforms within a single tandem cassette are shown as a black line above the gene structure diagram, while splice isoforms from different tandem cassettes are represented below by coloured lines. (d) Alternative splicing junctions from different cassettes were validated using reverse transcription–PCR (RT–PCR). Due to the low expression of sDscam variable exons, nested PCR was necessary to amplify the products; only the primers used in the second PCR are depicted and same in panels below. The RT–PCR products were confirmed by cloning and sequencing. These experiments revealed the splicing of multiple cassette variants from different tandem cassettes. (e) Splicing patterns of the 5′ variable exons in sDscamβ3. (f) RT–PCR was used to detect alternative splice isoforms in sDscamβ3. (g) A summary of several types of isoforms with distinct Ig numbers generated by alternative splicing.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4835542&req=5

f5: Highly complex combinations of sDscam 5′ variable exons.(a) Schematic diagram for splicing patterns of the 5′ variable exons. Symbols used are the same as in Fig. 1. Canonical splicing isoforms were joined in neighbouring junctions in variable cassettes, according to the previous ‘cap-proximal splicing' model19. Non-canonical splicing isoforms included: (I) splicing isoforms from the same cassette with either exon 2, 3 or 4 skipped; (II) splicing isoforms that contained variable exons from tandem cassettes; as well as (III) the isoforms that contained within-cassette introns. (b) Quantification of the canonical and non-canonical splicing isoforms. (c) Schematic diagram of the splicing patterns of the 5′ variable exons in M. martensii sDscamβ1. Splice isoforms within a single tandem cassette are shown as a black line above the gene structure diagram, while splice isoforms from different tandem cassettes are represented below by coloured lines. (d) Alternative splicing junctions from different cassettes were validated using reverse transcription–PCR (RT–PCR). Due to the low expression of sDscam variable exons, nested PCR was necessary to amplify the products; only the primers used in the second PCR are depicted and same in panels below. The RT–PCR products were confirmed by cloning and sequencing. These experiments revealed the splicing of multiple cassette variants from different tandem cassettes. (e) Splicing patterns of the 5′ variable exons in sDscamβ3. (f) RT–PCR was used to detect alternative splice isoforms in sDscamβ3. (g) A summary of several types of isoforms with distinct Ig numbers generated by alternative splicing.
Mentions: Inconsistent with the presence of a large first exon in the clustered Pcdh gene4, a cassette repeat composed of two or four exons was identified in the clustered sDscam gene. This raised the question of how these variable exons were combined into distinct mRNA isoforms, particularly because the exclusion or multiple inclusions of exons 2, 3 or 4 variants would not result in a frameshift. To explore this, we defined exon junctions based on a total of 0.7 billion RNA-seq reads from different tissues. At least 264 distinct exon junctions were detected, 249 of which were joined neighbouring junctions in single tandem cassettes. This suggests that most isoforms could be made through joining neighbouring junctions in variable cassette regions. Moreover, we detected a small fraction of isoforms from the same cassette with either exon 2, 3 and/or 4 skipped. In these cases, the variable exon skipping resulted in an incomplete Ig domain (that is, the sDscamβ6 variable exon 3.1) (Fig. 5a). This abnormal splicing is analogous to the skipping of Dscam exon 4 variants, which results in a partial Ig2 domain and is likely to be biologically relevant28. In addition, we detected other non-canonical splicing isoforms that contained variable exons from different tandem cassettes, as well as the isoforms containing within-cassette introns (Fig. 5a). Based on the exon junctions from the RNA-seq data, we estimated that ∼10–40% of isoforms resulted from non-canonical splicing in most sDscam genes, which showed differential expression in various tissues (Fig. 5a,b; Supplementary Fig. 10a,b). Taken together, these data indicate that sDscams have potentially complex splicing patterns at the 5′ variable regions.

Bottom Line: Furthermore, extraordinary isoform diversity has been generated through a combination of alternating promoter and alternative splicing.These sDscams have a high sequence similarity with Drosophila Dscam1, and share striking organizational resemblance to the 5' variable regions of vertebrate clustered Pcdhs.Hence, our findings have important implications for understanding the functional similarities between Drosophila Dscam1 and vertebrate Pcdhs, and may provide further mechanistic insights into the regulation of isoform diversity.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biochemistry, Innovation Center for Signaling Network, College of Life Sciences, Zhejiang University, Hangzhou, Zhejiang ZJ310058, China.

ABSTRACT
Drosophila Dscam1 (Down Syndrome Cell Adhesion Molecules) and vertebrate clustered protocadherins (Pcdhs) are two classic examples of the extraordinary isoform diversity from a single genomic locus. Dscam1 encodes 38,016 distinct isoforms via mutually exclusive splicing in D. melanogaster, while the vertebrate clustered Pcdhs utilize alternative promoters to generate isoform diversity. Here we reveal a shortened Dscam gene family with tandemly arrayed 5' cassettes in Chelicerata. These cassette repeats generally comprise two or four exons, corresponding to variable Immunoglobulin 7 (Ig7) or Ig7-8 domains of Drosophila Dscam1. Furthermore, extraordinary isoform diversity has been generated through a combination of alternating promoter and alternative splicing. These sDscams have a high sequence similarity with Drosophila Dscam1, and share striking organizational resemblance to the 5' variable regions of vertebrate clustered Pcdhs. Hence, our findings have important implications for understanding the functional similarities between Drosophila Dscam1 and vertebrate Pcdhs, and may provide further mechanistic insights into the regulation of isoform diversity.

No MeSH data available.


Related in: MedlinePlus