Vicente-Crespo Marta, Pascual Maya, Fernandez-Costa Juan M, Garcia-Lopez Amparo, Monferrer Lidón, Miranda M Eugenia, Zhou Lei, Artero Ruben D
Department of Genetics, University of Valencia, Valencia, Spain.
PLoS One. 2008 Feb 20;3(2):e1613. doi: 10.1371/journal.pone.0001613.
Muscleblind-like proteins (MBNL) have been involved in a developmental switch in the use of defined cassette exons. Such transition fails in the CTG repeat expansion disease myotonic dystrophy due, in part, to sequestration of MBNL proteins by CUG repeat RNA. Four protein isoforms (MblA-D) are coded by the unique Drosophila muscleblind gene.
METHODOLOGY/PRINCIPAL FINDINGS: We used evolutionary, genetic and cell culture approaches to study muscleblind (mbl) function in flies. The evolutionary study showed that the MblC protein isoform was readily conserved from nematods to Drosophila, which suggests that it performs the most ancestral muscleblind functions. Overexpression of MblC in the fly eye precursors led to an externally rough eye morphology. This phenotype was used in a genetic screen to identify five dominant suppressors and 13 dominant enhancers including Drosophila CUG-BP1 homolog aret, exon junction complex components tsunagi and Aly, and pro-apoptotic genes Traf1 and reaper. We further investigated Muscleblind implication in apoptosis and splicing regulation. We found missplicing of troponin T in muscleblind mutant pupae and confirmed Muscleblind ability to regulate mouse fast skeletal muscle Troponin T (TnnT3) minigene splicing in human HEK cells. MblC overexpression in the wing imaginal disc activated apoptosis in a spatially restricted manner. Bioinformatics analysis identified a conserved FKRP motif, weakly resembling a sumoylation target site, in the MblC-specific sequence. Site-directed mutagenesis of the motif revealed no change in activity of mutant MblC on TnnT3 minigene splicing or aberrant binding to CUG repeat RNA, but altered the ability of the protein to form perinuclear aggregates and enhanced cell death-inducing activity of MblC overexpression.
CONCLUSIONS/SIGNIFICANCE: Taken together our genetic approach identify cellular processes influenced by Muscleblind function, whereas in vivo and cell culture experiments define Drosophila troponin T as a new Muscleblind target, reveal a potential involvement of MblC in programmed cell death and recognize the FKRP motif as a putative regulator of MblC function and/or subcellular location in the cell.
类肌肉盲蛋白（MBNL）参与了特定盒式外显子使用过程中的发育转换。在CTG重复扩增疾病强直性肌营养不良中，这种转换失败，部分原因是CUG重复RNA隔离了MBNL蛋白。果蝇独特的肌肉盲基因编码四种蛋白质异构体（MblA - D）。
方法/主要发现：我们使用进化、遗传和细胞培养方法来研究果蝇中肌肉盲（mbl）的功能。进化研究表明，从线虫到果蝇，MblC蛋白质异构体很容易保守下来，这表明它执行最原始的肌肉盲功能。在果蝇眼祖细胞中过表达MblC会导致眼睛外观粗糙。这种表型被用于遗传筛选，以鉴定五个显性抑制因子和13个显性增强因子，包括果蝇CUG - BP1同源物aret、外显子连接复合体成分tsunagi和Aly，以及促凋亡基因Traf1和reaper。我们进一步研究了肌肉盲在凋亡和剪接调控中的作用。我们在肌肉盲突变蛹中发现肌钙蛋白T的错配剪接，并证实肌肉盲能够在人HEK细胞中调节小鼠快骨骼肌肌钙蛋白T（TnnT3）小基因的剪接。在翅成虫盘过表达MblC以空间受限的方式激活凋亡。生物信息学分析在MblC特异性序列中鉴定出一个保守的FKRP基序，与一个类SUMO化靶位点微弱相似。对该基序进行定点诱变显示，突变的MblC对TnnT3小基因剪接的活性或与CUG重复RNA的异常结合没有变化，但改变了蛋白质形成核周聚集体的能力，并增强了过表达MblC诱导细胞死亡的活性。
结论/意义：综合来看，我们的遗传方法确定了受肌肉盲功能影响的细胞过程，而体内和细胞培养实验将果蝇肌钙蛋白T定义为一个新的肌肉盲靶点，揭示了MblC可能参与程序性细胞死亡，并将FKRP基序识别为MblC功能和/或细胞内亚细胞定位的假定调节因子。
