異質核核糖核蛋白(Heterogeneous nuclear ribonucleoproteins, hnRNPs)在pre-mRNA熟成(maturation)的眾多過程中，扮演著相當重要的角色，例如RNA的包裹作用(RNA packaging)與選擇性剪接作用(alternative splicing)，因此不論是存在於線蟲體內或是哺乳動物的hnRNPs，其在基因的序列上皆具有極高的保守性。果蠅hnRNP A1型同源基因- hrp36能參與並調控許多基因的選擇性剪接作用，其中最著名的例子為Down syndrome cell adhesion molecule(Dscam)，其可以從單一基因座產生萬種的異構型(isofroms)。在本篇研究中，成功的自白蝦(Litopenaeus vannamei)中鑑定出第一個蝦類hrp36同源基因- Lvhrp36。Lvhrp36的開放譯讀區(open reading fram, ORF)長1,101 bp，預計編碼出366個胺基酸。由胺基酸序列所預測出的蛋白質結構中含有兩組RNA辨識模體(RNA recognition motif, RRM)、C端(C-terminal)富含甘胺酸結構域(glycine-rich domain, GRD)、高度退化性的RNP保留序列RNP-1與RNP-2，另外還具有RGG boxes。經由組織特異性的分析顯示Lvhrp36廣泛性的存在於白蝦體內，且在大部分的組織與器官具有高度的表現量。藉由在白蝦活體內注射雙股RNA將Lvhrp36沉默化，會誘導LvDscam發生異常性的外顯子(Exon)剪接，這個現象在Ig3變異區更為顯著。在LvDscam Ig3的部分，我們共發現了14種不同形式的異構型組合，分別來自3種不同的異常性剪接模式。此外，在Ig2也發現1種異構型組合(來自1種異常性剪接模式)；Ig7則是發現3種異構型組合(來自1種異常性剪接模式)。這是目前所發現的第一項證據顯示hrp36可能也參與在Ig7變異區的調控。值得注意的是，至少目前在果蠅的Ig7外顯子群(Ig7 exon cluster)中並不具有hrp36的結合序列。然而，上述的異常性剪接現象無法在每隻經過Lvhrp36沉默化處理的白蝦個體中被觀察到。即使有異常性剪接的現象發生，正常剪接形式的LvDscam仍然較為常見。我們推測上述情況可能是由於其他沒被沉默化且可以替代Lvhrp36功能的蛋白質所造成。綜觀而言，我們的結果顯示Lvhrp36的功能可能作為剪接作用的阻遏子(repressor)，藉以調控白蝦L. vannamei Dscam Ig2、Ig3與Ig7變異區的選擇性剪接作用。

Heterogeneous nuclear ribonucleoproteins (hnRNPs) are highly conserved from nematode to mammal because they play an important role in several aspects of pre-mRNA maturation, including RNA packaging and alternative splicing. In Drosophila, the hnRNP A1 homolog hrp36 regulates alternative splicing in several genes, including the Down syndrome cell adhesion molecule (Dscam), which produces tens of thousands of isoforms from one locus. In this study, the first hrp36 gene was identified and characterized from Litopenaeus vannamei (Lvhrp36). Its open reading frame (ORF) contains 1,101 bp encoding 366 amino acids. The deduced Lvhrp36 protein includes two copies of the RNA recognition motif (RRM), a C-terminal glycine-rich domain (GRD), the highly degenerate RNP consensus sequences RNP-1 and RNP-2, and two RGG boxes. Tissue tropism analysis indicated that Lvhrp36 is expressed ubiquitously and at high levels in most tissues. dsRNA silencing of shrimp Lvhrp36 in vivo induced abnormal exon inclusions in LvDscam, especially in the Ig3 variable region. In the Ig3 region, a total of 14 different combinations were arranged in three different types of abnormal inclusion pattern. This compares to a single combination (one abnormal pattern) in Ig2 and three different combinations (one abnormal pattern) in Ig7. This is the first evidence to suggest that hrp36 may be involved in the regulation of the Ig7 variable region, and it is noteworthy because, at least in Drosophila, there are no hrp36 binding sequences in the Ig7 exon cluster. The above aberrant events were not observed in all of the Lvhrp36-silenced shrimp, and even when they occurred, the normal patterns of inclusion were far more common. We hypothesize that this continued prevalence of normal inclusions was probably due to other unsilenced proteins that were able to rescue Lvhrp36’s functionality. Taken together, our results suggest that Lvhrp36 acts as a splicing repressor that regulates alternative splicing events in the Ig2, Ig3 and Ig7 variable regions of shrimp L. vannamei Dscam.

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