Molecular characterization of two splicing-related genes, SPF30 and SR30, in Arabidopsis

Alternative splicing (AS) is an important post-transcriptional regulation mechanism in eukaryotes that can generate multiple mRNA transcripts from a single DNA template. Splicing is executed by spliceosome, and the process is assisted and regulated by numerous protein factors. Recent high-throughput transcriptome analyses have revealed that AS is a prevalent phenomenon in plants. In addition, many splicing factor genes themselves are alternatively spliced, but the roles of most identified splice isoforms remain unclear. To gain insight into the splicing regulation in plants, we investigated two splicing-related genes that encode for splicing factor 30 (SPF30) and serine/arginine-rich protein 30 (SR30), respectively, in Arabidopsis thaliana. Mammalian orthologs of SPF30 and SR30 have been well characterized as essential splicing factors, whereas the research on these two proteins in plants has been limited.In this thesis we have shown that plant SPF30 family genes undergo conserved AS events associated with their second introns. These AS events often cause premature termination but occasionally lead to the birth of new exons. In Arabidopsis, SPF30 has three transcript isoforms with different coding sequences. Particularly, SPF30.1 localized to the cell nucleus, while SPF30.3 diffused throughout the cell. Moreover, we identified an SPF30 knockout mutant (spf30) that displayed early flowering phenotype and reduced level of FLOWERING LOCUS C (FLC), a key regulator of flowering time. Similar to SPF30, SR30 undergoes AS and its protein isoforms have different subcellular localization patterns. Interestingly, SR30.4 displayed a novel localization in the peroxisome due to acquisition of a peroxisome targeting signal 1 (PTS1) motif. When expressed in Arabidopsis protoplasts, all five SR30 isoforms co-localized with spliceosomal protein U1-70K in distinct nuclear speckles. Overexpression of SR30.2-5 in transgenic Arabidopsis had different effects on the AS of endogenous SR30 compared to overexpression of SR30.1. PCR analysis indicates that the expression and AS of SR30 was responsive to multiples environmental signals, such as light, dark and heat. Furthermore, transcriptome analysis has revealed that the AS of a wide range of genes, including splicing-related genes and circadian clock genes, was affected in sr30. However, sr30, or other SR subfamily gene mutants, sr34 and sr34b, did not exhibit obvious phenotypes under normal growth conditions, suggesting some degree of functional redundancy. Taken together, this work reports the biological functions of SPF30 and SR30 in Arabidopsis and reveals the different properties of the splice variants encoded by these two genes.可變剪接是真核生物中的一種重要的轉錄後調控方式，該機制可以從單個DNA 模版中產生多個不同的mRNA 轉錄本。剪接過程是由剪接復合體執行的，同時也受到眾多蛋白因子的輔助和調控。近年來的高通量轉錄組分析揭示植物中的可變剪接現象十分普遍，甚至許多編碼剪接因子的基因自身也發生可變剪接，但是大部分已經鑒定到的可變剪接本的功能尚不清晰。為了深入瞭解植物中的剪接調控，本研究圍繞模式植物擬南芥的兩個剪接相關基因SPF30 和SR30 展開。這兩個基因編碼的蛋白在哺乳動物中的同源蛋白都是至關重要的剪接因子，且已經被研究得較為詳細，而在植物中的研究卻十分有限。本研究發現植物的SPF30 家族基因的第二個內含子發生保守的可變剪接事件。這些可變剪接事件經常引起編碼區提前終止，但偶爾也會導致新外顯子誕生。在擬南芥中，SPF30 基因具有三條含不同編碼序列的轉錄本。其中SPF30.1 蛋白定位在細胞核，而SPF30.3 蛋白則彌散在整個細胞內。本研究鑒定到了一個SPF30 基因的敲除突變體（spf30），該突變體表現出提早開花的表型，而且調節開花時間的一個關鍵基因FLC 在該突變體中的mRNA 含量有所降低。類似SPF30，SR30 基因也發生自身的可變剪接，且因此產生的蛋白異構體具有不同的亞細胞定位模式。有趣的是，SR30.4 蛋白的序列里隱含了一個1 型過氧化物酶體定位信號肽，並因此定位在了過氧化物酶體中。當在擬南芥原生質體里共表達時，5 個SR30 的蛋白異構體全都可以和剪接復合體蛋白U1-70K 共同定位到核斑點中。而在穩定轉化的擬南芥中過量表達SR30.2-5 時，它們對內源SR30 基因的可變剪接產生的效應與過表達SR30.1 不同。PCR 分析表明SR30 基因的表達和可變剪接會受到多種環境信號的影響，比如光暗和熱。通過轉錄組分析發現在SR30 的突變體中，大量基因的可變剪接模式受到影響，其中包括剪接相關基因以及生物鐘相關基因。不過，本研究中並未觀察到SR30 的突變體，或是SR30 的同源基因SR34 和SR34b的突變體在常規生長條件下的差異表型，提示這個家族的基因存在一定程度的功能冗余。綜上所述，本論文報道了SPF30 和SR30 在擬南芥中的生物學功能，並揭示了這兩個基因所編碼的不同轉錄本的分子特性。Zhang, Di.Ph.D. Chinese University of Hong Kong 2020.Includes bibliographical references (leaves 114-124).Abstracts also in Chinese.Title from PDF title page (viewed on October 14, 2022)