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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

The retention of intron sequences immediately downstream of the last variable exon of each cassette.(a) Schematic diagram of sDscamβ1 isoform expression. Symbols used are the same as in Figs 1 and 4. The expression of the specific combination of sDscam isoforms was achieved by alternative promoter activation, followed by alternative splicing. When sDscamβ1 was transcribed by a V5 promoter, both V5 and the downstream V6 cassette may have been spliced into the constant exon 5. The positions of the PCR primers are indicated. (b) Intron retention downstream of the 5′ splice site of the variable cassette (V5) in sDscamβ1 mRNA reads. Intron retention was much more abundant in the abdomen than in the cephalothorax. The 25-nt fragmented RNA-seq data sets were mapped to calculate the intron retention rate. Because of the low expression of the V5 and V6 isoform in the muscles, haemocytes and poison glands (Fig. 3c), the images of these RNA-seq reads are not shown. (c) RT–PCR analysis of V5 and V6 isoform expression. (d) Schematic diagrams of expression of sDscamβ2 isoforms. Different types of splice isoforms are indicated by the symbol "I, II, III, IV". (e) RT–PCR was used to detect isoform expression. These experiments revealed the splicing of multiple adjacent cassette variants. 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. The PCR products were confirmed by cloning and sequencing.
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f6: The retention of intron sequences immediately downstream of the last variable exon of each cassette.(a) Schematic diagram of sDscamβ1 isoform expression. Symbols used are the same as in Figs 1 and 4. The expression of the specific combination of sDscam isoforms was achieved by alternative promoter activation, followed by alternative splicing. When sDscamβ1 was transcribed by a V5 promoter, both V5 and the downstream V6 cassette may have been spliced into the constant exon 5. The positions of the PCR primers are indicated. (b) Intron retention downstream of the 5′ splice site of the variable cassette (V5) in sDscamβ1 mRNA reads. Intron retention was much more abundant in the abdomen than in the cephalothorax. The 25-nt fragmented RNA-seq data sets were mapped to calculate the intron retention rate. Because of the low expression of the V5 and V6 isoform in the muscles, haemocytes and poison glands (Fig. 3c), the images of these RNA-seq reads are not shown. (c) RT–PCR analysis of V5 and V6 isoform expression. (d) Schematic diagrams of expression of sDscamβ2 isoforms. Different types of splice isoforms are indicated by the symbol "I, II, III, IV". (e) RT–PCR was used to detect isoform expression. These experiments revealed the splicing of multiple adjacent cassette variants. 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. The PCR products were confirmed by cloning and sequencing.

Mentions: Finally, we examined how variable exons were spliced after transcription by alternative promoters. Although previous studies suggested that only the cap-proximal variable exon was joined to the first constant exon in vertebrate Pcdhs41920, this hypothesis had not been validated experimentally due to the large size (∼200 kb in the variable regions) and complexity of the clustered Pcdhs. Surprisingly, we found that abundant intron sequences immediately downstream of the last variable exon of each cassette were frequently retained in the RNA-seq data, while cassettes within introns were exclusively spliced out (that is, sDscamβ1 V5, Fig. 6a,b). Interestingly, the extent of this retention differed in different tissues (Fig. 6b; Supplementary Fig. 10b). The frequent occurrence of this unusual intronic retention might be a result of the splicing of the variable exons immediately downstream of the cap-proximal cassette to the constant exon (type II; Fig. 6a). Taken together, we propose that not only the cap-proximal, but also the downstream variable exons spliced to the constant exon.


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)

The retention of intron sequences immediately downstream of the last variable exon of each cassette.(a) Schematic diagram of sDscamβ1 isoform expression. Symbols used are the same as in Figs 1 and 4. The expression of the specific combination of sDscam isoforms was achieved by alternative promoter activation, followed by alternative splicing. When sDscamβ1 was transcribed by a V5 promoter, both V5 and the downstream V6 cassette may have been spliced into the constant exon 5. The positions of the PCR primers are indicated. (b) Intron retention downstream of the 5′ splice site of the variable cassette (V5) in sDscamβ1 mRNA reads. Intron retention was much more abundant in the abdomen than in the cephalothorax. The 25-nt fragmented RNA-seq data sets were mapped to calculate the intron retention rate. Because of the low expression of the V5 and V6 isoform in the muscles, haemocytes and poison glands (Fig. 3c), the images of these RNA-seq reads are not shown. (c) RT–PCR analysis of V5 and V6 isoform expression. (d) Schematic diagrams of expression of sDscamβ2 isoforms. Different types of splice isoforms are indicated by the symbol "I, II, III, IV". (e) RT–PCR was used to detect isoform expression. These experiments revealed the splicing of multiple adjacent cassette variants. 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. The PCR products were confirmed by cloning and sequencing.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f6: The retention of intron sequences immediately downstream of the last variable exon of each cassette.(a) Schematic diagram of sDscamβ1 isoform expression. Symbols used are the same as in Figs 1 and 4. The expression of the specific combination of sDscam isoforms was achieved by alternative promoter activation, followed by alternative splicing. When sDscamβ1 was transcribed by a V5 promoter, both V5 and the downstream V6 cassette may have been spliced into the constant exon 5. The positions of the PCR primers are indicated. (b) Intron retention downstream of the 5′ splice site of the variable cassette (V5) in sDscamβ1 mRNA reads. Intron retention was much more abundant in the abdomen than in the cephalothorax. The 25-nt fragmented RNA-seq data sets were mapped to calculate the intron retention rate. Because of the low expression of the V5 and V6 isoform in the muscles, haemocytes and poison glands (Fig. 3c), the images of these RNA-seq reads are not shown. (c) RT–PCR analysis of V5 and V6 isoform expression. (d) Schematic diagrams of expression of sDscamβ2 isoforms. Different types of splice isoforms are indicated by the symbol "I, II, III, IV". (e) RT–PCR was used to detect isoform expression. These experiments revealed the splicing of multiple adjacent cassette variants. 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. The PCR products were confirmed by cloning and sequencing.
Mentions: Finally, we examined how variable exons were spliced after transcription by alternative promoters. Although previous studies suggested that only the cap-proximal variable exon was joined to the first constant exon in vertebrate Pcdhs41920, this hypothesis had not been validated experimentally due to the large size (∼200 kb in the variable regions) and complexity of the clustered Pcdhs. Surprisingly, we found that abundant intron sequences immediately downstream of the last variable exon of each cassette were frequently retained in the RNA-seq data, while cassettes within introns were exclusively spliced out (that is, sDscamβ1 V5, Fig. 6a,b). Interestingly, the extent of this retention differed in different tissues (Fig. 6b; Supplementary Fig. 10b). The frequent occurrence of this unusual intronic retention might be a result of the splicing of the variable exons immediately downstream of the cap-proximal cassette to the constant exon (type II; Fig. 6a). Taken together, we propose that not only the cap-proximal, but also the downstream variable exons spliced to the constant exon.

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