Journal: Science Advances

Article Title: Trans-splicing facilitated by RNA pairing greatly expands sDscam isoform diversity but not homophilic binding specificity

doi: 10.1126/sciadv.abn9458

Figure Lengend Snippet: See also figs. S1 and S2. ( A ) Phylogenetic distribution of sDscam and isoform members in chelicerates. The variables Ig1s and Ig2s are indicated by green and red circles, respectively. Data from other species are referenced from our previous study . ( B ) Schematic of an sDscam locus. The 5′ untranslated region of sDscam β 4 is represented by a gray rectangle. The arrow indicates transcriptional direction. Cis- and trans-spliced isoforms are represented by blue lines (above) and other colored lines (below), respectively. The color connections are supported by RNA-seq and RT-PCR data. Var, variable; Con (C), constant. ( C ) Quantification of the cis- and trans-spliced isoforms. RPM, reads per million. ( D ) Validation of alternative combinations of 5′ and 3′ alternative exons. Because of the low expression of variable exons, nested PCR was required to amplify the products; only the primers used in the second PCR are depicted (table S2). ( E to J ) Evidence of trans-splicing between different genes. These combinations included sDscam β 2 and sDscam β 1 (E), sDscam β 3 and sDscam β 1 (F), sDscam β 2 –β3 and sDscam β 4 (G), sDscam β 1 / sDscam β 3 and sDscam β 2 (H), sDscam β 1 and sDscam β 3 (I), and sDscam β 2 and sDscam β 3 (J).

Article Snippet: The primary antibodies were used in isoform coimmunoprecipitation: anti-HA (hemagglutinin) tag rabbit polyclonal antibody (1:50; EarthOx, catalog no. E022180-01, RRID:AB_2811272).

Techniques: RNA Sequencing Assay, Reverse Transcription Polymerase Chain Reaction, Expressing, Nested PCR

Journal: Science Advances

Article Title: Trans-splicing facilitated by RNA pairing greatly expands sDscam isoform diversity but not homophilic binding specificity

doi: 10.1126/sciadv.abn9458

Figure Lengend Snippet: See also fig. S12. ( A and B ) Domain-specific recognition of the N-variable mediated by the sDscamβ Ig domain shuffled isoform. Domain-shuffled chimeras of sDscamβ isoforms and their parental counterparts were assayed for their binding specificity. Chimeras in which the Ig1 domain was replaced by the corresponding domain swapped binding specificity, whereas the Ig2 replacement did not. ( C ) sDscamβ1 pairs with the same variable Ig1 domain do not display recognition specificity. Mean coaggregation indices are shown in the top right corner of each representative image (scale bars, 100 μm). ( D ) Schematic diagram of trans interactions of sDscamβ. Structural modeling shows that the Ig1 domain of sDscamβ interacts in an antiparallel manner.

Article Snippet: The primary antibodies were used in isoform coimmunoprecipitation: anti-HA (hemagglutinin) tag rabbit polyclonal antibody (1:50; EarthOx, catalog no. E022180-01, RRID:AB_2811272).

Techniques: Binding Assay

Journal: Science Advances

Article Title: Trans-splicing facilitated by RNA pairing greatly expands sDscam isoform diversity but not homophilic binding specificity

doi: 10.1126/sciadv.abn9458

Figure Lengend Snippet: See also fig. S13. ( A to D ) Cells coexpressing different combinations of differentially tagged sDscamβ isoform pairs were mixed and assayed for their coaggregation. β1 cis-spliced isoforms (A), β1 trans-spliced isoforms (B), β1/β2 cis-spliced isoforms (C), and β1/β2 trans-spliced isoforms (D) were measured. ( E ) The combination of cis- and trans-spliced sDscamβ pairs with the same variable Ig1-Ig2 domains did not exhibit the combinatorial homophilic specificity. ( F ) Analysis of the interaction of cells coexpressing three different GFP tags with cells expressing the same or different groups of mCherry tags. The underline marks the mismatched isoforms between the two cell groups. Mean coaggregation indices for (A) to (F) are shown in the top right corner of each representative image (scale bars, 100 μm). ( G ) Schematic diagram of the outcome of combinatorial homophilic specificity. The diagram shown here does not reflect cis multimers.

Article Snippet: The primary antibodies were used in isoform coimmunoprecipitation: anti-HA (hemagglutinin) tag rabbit polyclonal antibody (1:50; EarthOx, catalog no. E022180-01, RRID:AB_2811272).

Techniques: Expressing

Journal: Science Advances

Article Title: Trans-splicing facilitated by RNA pairing greatly expands sDscam isoform diversity but not homophilic binding specificity

doi: 10.1126/sciadv.abn9458

Figure Lengend Snippet: ( A ) Generation of extensive sDscam isoforms through a combination of alternative promoter choices and cis- and trans-alternative splicing. On the left is the schematic representation generating cis-spliced sDscamβ isoform diversity. This alternative cis-splicing process is mediated by a competing RNA secondary structure between the docking site and selector sequences. On the right is a schematic representation of the generation of trans-spliced sDscamβ isoforms. This trans-splicing process is facilitated by intronic intermolecular RNA secondary structures. ( B ) Schematic representation of sDscamβ diversity mediated by cotranscriptional RNA folding and alternative cis- and trans-splicing. These nascent transcripts generated from this single locus are geometrically close to each other before leaving their transcription sites, which facilitates trans-splicing between different sDscam β transcripts.

Article Snippet: The primary antibodies were used in isoform coimmunoprecipitation: anti-HA (hemagglutinin) tag rabbit polyclonal antibody (1:50; EarthOx, catalog no. E022180-01, RRID:AB_2811272).

Techniques: Generated