Alternatively spliced BobCAL transcripts alter curd morphotypes in a collection of Chinese cauliflower accessions

The curd of cauliflower(Brassica oleracea L.var.botrytis)is a modified inflorescence that is consumed as a vegetable.Curd formation is proposed to be due to a mutation in the BobCAULIFLOWER(BobCAL)gene,but the genetic relationship between BobCAL variation and curd morphotypes remains obscure.To address this question,we collected and classified a collection of 78 cauliflower accessions into four subpopulations according to curd surface features:smooth,coarse,granular,and hairy curd morphotypes.Through the cDNA sequencing of BobCAL alleles,we showed that smooth and coarse accessions characterized by inflorescence meristem arrest presented a strong association with the 451T SNP(BobCAL_T),whereas granular and hairy accessions marked with floral organ arrest presented an association with 451G(BobCAL_G).Interestingly,all BobCAL alleles were alternatively spliced,resulting in a total of four alternative splice(AS)variants due to the retention of the fourth and/or seventh introns.Among accessions with BobCAL_G alleles,the total expression of all these AS variants in granular plants was almost equal to that in hairy plants;however,the expression of the individual AS variants encoding intact proteins relative to those encoding truncated proteins differed.Hairy accessions showed relatively high expression of the individual variants encoding intact proteins,whereas granular accessions displayed relatively low expression.In smooth cauliflower,the overexpression of the BobCAL_Ga variant caused an alteration in the curd morphotype from smooth to hairy,concurrent with an increase in the expression levels of downstream floral identity genes.These results reveal that alternative splicing of BobCAL transcripts is involved in the determination of cauliflower curd morphotypes.

HITS-CLIP reveals sex-differential RNA binding and alterative splicing regulation of SRm160 in Drosophila

Serine/arginine (SR)-rich proteins are critical for the regulation of alternative splicing (AS), which generates multiple mRNA isoforms from one gene and provides protein diversity for cell differentiation and tissue development. Genetic evidence suggests that Drosophila genital-specific overexpression of SR-related nuclear matrix protein of 160 kDa (SRml60), an SR prot&n with a PWI RNA-binding motif, causes defective development only in male flies and results in abnonnal male genital structures and abnormal testis. However, the molecular characterization of SRm160 is limited. Using the high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP) method in two sex-specific embryonic cell lines, S2 from the male and Kc from the female, we first identified the genome-wide RNA-binding characteristics of SRm160, which preferred binding to the exonic tri-nucleotide repeats GCA and AAC. We then validated this binding through both in vitro gel-shift assay and in vivo splicing of minigenes and found that SRm160 level affects AS of many transcripts. Furthermore, we identified 492 differential binding sites (DBS) of SRm160 varying between the two sex-specific cell lines.Among these DBS-containing genes, splicing factors were highly enriched, including transformer, a key regulator in the sex determination cascade. Analyses of fly mutants demonstrated that the SRm160 level affects AS isoforms of transformer. These findings shed crucial light on SRm160's RNA-binding specificity and regulation of AS in Drosophila sex determination and development.