Muscleblind 蛋白質於斑馬魚早期發育時的功能性分析

Muscleblind(MBNL)家族蛋白質為一種參與組織特異性RNA差異剪輯的調節蛋白。其蛋白質家族共同的特徵在於具有CCCH zinc finger domin，並藉由此結合RNA。MBNL功能異常已知會造成人類肌強直肌肉萎縮症。本篇研究於斑馬魚中選殖兩個muscleblind 基因：z-mbnl1以及z-mbnl2，其RNA差異剪輯一共可編碼至少8種蛋白質異構物。除了最具特徵的zinc fingers之外，mbnl蛋白質還具有數個其他結構區域，包括一個C端的Ser/Thr-rich region，這些區域在其他脊椎動物的mbnl蛋白質中也有序列的保留。Mbnl基因廣泛表現於斑馬魚的成魚組織中，只是其特定的剪輯形式於各種組織表現上具有差異性。在斑馬魚胚胎早期便有來自母源的z-mbnl1和z-mbnl2基因的表現，直至胚胎發育時期各基因型的表現量也明顯不同。我們利用全胚胎原位雜交(whole mount in situ hybridyzation)觀察mbnl在不同時間與空間上的表現情形。z-mbnl1在胚胎發育過程中表現於血液前趨細胞(presumptive blood)、腹側中胚層、腸道、肛門、心臟、中/後腦交界、肝臟、魚鰾、耳囊及咽喉等組織。z-mbnl2的表現則分部於眼睛、嗅覺上皮、鰓弓、頭部間葉組織、視神經叉、中/後腦交界、咽喉及魚鰾組織中。以顯微注射 morpholino來抑制z-mbnl1及z-mbnl2 mRNA的表現，發現z-mbnl1 knockdown的胚胎，有中/後腦交界變短、眼睛分化不全、胚胎不易自然脫出卵膜、心臟發育異常、血液循環緩慢、下顎和魚鰾缺失、耳石融合及腹腔水腫等。而注射z-mbnl2 morphant的胚胎，其表型均與z-mbnl1 knockdown的胚胎相似，但其嚴重程度卻較為輕微。使用自製z-mbnl1及z-mbnl2抗體以西方墨點法偵測蛋白表現量，可觀察到z-mbnl1或z-mbnl2蛋白在morpholino knockdown 的胚胎中其蛋白量有明顯下降。並且以coinjection分別加入z-mbnl1及z-mbnl2 訊息RNA，統計結果發現可部分回覆上述異常表型。以上兩點證明morpholino之效力與專一性。此外，以半定量RT-PCR觀察，發現z-mbnl1對於下游基因tnnt2 、mtmr1、vinculin以及clcn1的splicing有異常的調節，而z-mbnl2的缺失對clcn1 gene的splicing似乎沒有明顯的影響。相對於DM病人中肌肉無力與心臟功能失常的病徵，z-mbnl1或者z-mbnl2的缺少使得這些與肌肉及心臟相關基因的特異性剪接異常，可能是造成斑馬魚胚胎無法自行脫出卵膜及心臟發育異常的原因。而z-mbnl2的缺失所造成的嚴重程度較z-mbnl1的缺少來得輕微，顯示z-mbnl1在發育期間所扮演的功能角色，可能比z-mbnl2更為重要。已有文獻顯示z-mbnl1可調控早期肌肉分化的進行，至於其中的調控機制則有待於更進一步的實驗分析。

關鍵字

班馬魚；全胚胎原位雜交；組織特異性RNA差異剪輯

並列摘要

Muscleblind (MBNL) is a family of proteins that participate in regulation of tissue-specific alternative splicing. They bind to RNA through a conserved CCCH zinc finger domain. Misregulation of MBNL activity in human leads to pathogenesis. We have cloned three muscleblind genes in zebrafish (zmbnl1, zmbnl2 and zmbnl3). Alternative splicing of the two zmbnl primary transcripts gives rise to at least 13 protein isoforms. In addition to the characteristic zinc fingers, several structural motifs, including a C-terminal Ser/Thr-rich region, are found conserved among vertebrate mbnl orthologs. These genes are broadly expressed in most adult tissues although the expression of specific spliceforms varies across different tissues. During embryogenesis, zmbnl1 and zmbnl2 are both maternally and zygotically expressed. Whole-mount in situ hybridization reveals that zmbnl1 is expressed in lens, liver, otic vesicle, muscle and pharynx, while zmbnl2 is expressed in lens, olfactory epithelium, branchial arches, head mesenchymes, optic chiasm, midbrain hindbrain boundary, pharynx and swim bladder. Knockdown of zmbnl1 and zmbnl2 results in morphants with deformed brain, eyes, otoliths, heart and pharyngeal arches. In addition, these morphants are defective in hatching and swimming behavior. In consistent with the phenotypes, the splicing patterns of four pre-mRNAs (tnnt2, mtmr1,clcn1 and vinculin) that are misregulated in cells with CUG RNA expansion, are altered in the zmbnl morphants. These data suggest that zebrafish MBNL proteins are crucial for early fish development possibly through the regulation of specific gene splicing.

並列關鍵字

zebrafish ； muscleblind ； splicing regulator ； whole mount in situ hybridyzation

參考文獻

1. Faustino, N.A. and T.A. Cooper, Pre-mRNA splicing and human disease. Genes Dev, 2003. 17(4): p. 419-37.

2. Pascual, M., et al., The Muscleblind family of proteins: an emerging class of regulators of developmentally programmed alternative splicing. Differentiation, 2006. 74(2-3): p. 65-80.

3. Ho, T.H., et al., Muscleblind proteins regulate alternative splicing. EMBO J, 2004. 23(15): p. 3103-12.

4. Worthington, M.T., et al., Metal binding properties and secondary structure of the zinc-binding domain of Nup475. Proc Natl Acad Sci U S A, 1996. 93(24): p. 13754-9.

5. Lai, W.S., E.A. Kennington, and P.J. Blackshear, Interactions of CCCH zinc finger proteins with mRNA: non-binding tristetraprolin mutants exert an inhibitory effect on degradation of AU-rich element-containing mRNAs. J Biol Chem, 2002. 277(11): p. 9606-13.

國際替代計量

Muscleblind 蛋白質於斑馬魚早期發育時的功能性分析

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