Crosstalk between vrille transcripts, proteins and regulatory elements controlling circadian rhythms and development in Drosophila

The vrille (vri) gene encodes a bZIP transcriptional repressor that is required for Drosophila patterning and cell proliferation during development as well as circadian behavior in adults. Three alternate first exons produce five different vri transcripts, where first exons 1a and 1b initiate two transcripts during development that produce the 610aa short VRI isoform and first exon 1c initiates three transcripts in adults that produce the 729aa long VRI isoform. To test whether long VRI is necessary for circadian clock function, we generated a mutant (vri?679) that eliminates transcripts encoding long VRI. The vri?679 mutant is viable, confirming that developmental functions of vri are driven by transcripts from exons 1a/1b, yet behavioral rhythms persist in vri?679 flies, demonstrating that vri transcripts encoding short VRI are sufficient for clock output. E-box regulatory elements upstream of exon 1c that drive rhythmic transcription in adults are also required for development, implying that these E-boxes also drive vri transcription from exons 1a/1b. Surprisingly, there is almost no long VRI present in adults even though the vast majority of transcripts encode long VRI, apparently because the ATG start codon for long VRI has a poor Kozak sequence context. Consequently, short VRI is the predominant isoform in adults and is sufficient for clock output. We conclude that vri-ADF transcripts driven by E-boxes upstream of exon 1c primarily control circadian rhythms by producing short VRI , whereas vri-E transcripts that are also regulated by E-boxes upstream of exon 1c mediate developmental patterning/cell proliferation through short VRI. Overall design: RNA-seq: mRNA profiles of heads from Drosophila wild type (WT) and vri?679 mutant at two different timepoints, ZT 02 and ZT 14

vrille（vri）基因编码一种碱性亮氨酸拉链（bZIP）转录抑制因子，该因子对于果蝇发育过程中的模式构建、细胞增殖，以及成虫的昼夜节律行为均不可或缺。三个可变第一外显子可产生五种不同的vri转录本：其中外显子1a与1b在发育过程中启动两种转录本，编码610个氨基酸的短型VRI同工型；外显子1c则在成虫体内启动三种转录本，编码729个氨基酸的长型VRI同工型。为探究长型VRI对于昼夜节律时钟功能是否必需，我们构建了vri?679突变体，该突变体可剔除编码长型VRI的转录本。vri?679突变体可存活，这证实vri的发育功能由外显子1a/1b来源的转录本所介导；而vri?679果蝇仍保留行为节律，表明编码短型VRI的vri转录本足以支撑节律输出。外显子1c上游的E盒（E-box）调控元件可驱动成虫体内的节律性转录，同时也对发育过程不可或缺，这暗示此类E盒同样可驱动外显子1a/1b来源的vri转录。令人意外的是，尽管绝大多数转录本均编码长型VRI，但成虫体内几乎检测不到长型VRI，这显然是因为长型VRI的ATG起始密码子所处的科扎克序列（Kozak sequence）环境不佳。因此，短型VRI是成虫体内的主要同工型，且足以支撑节律输出。综上，由外显子1c上游E盒调控的vri-ADF转录本主要通过生成短型VRI来调控昼夜节律；而同样受外显子1c上游E盒调控的vri-E转录本则通过短型VRI介导发育模式构建与细胞增殖。实验整体设计：RNA测序（RNA-seq）：采集果蝇野生型（WT）与vri?679突变体头部的mRNA表达谱，采样时间点为ZT 02与ZT 14