來自麻省理工學(xué)院，Whitehead生物醫(yī)藥研究所，霍德華休斯醫(yī)學(xué)院的研究人員提出了一種新的轉(zhuǎn)錄組測(cè)序高通量方法，這種方法能避免“poly（A）”方法的局限性，對(duì)于轉(zhuǎn)錄組測(cè)序方法的發(fā)展具有重要意義。這一研究成果公布在Nature雜志上。

領(lǐng)導(dǎo)這一研究的是麻省理工學(xué)院的David P. Bar教授，這位microRNA方面*的人物湯姆森科技信息集團(tuán)公布的“生物學(xué)領(lǐng)域zui熱門的機(jī)構(gòu)、作者、期刊排名”中，入選高影響力論文數(shù)量排名作者：Bar教授的19篇高影響力論文共被引4542次，每篇論文平均被引239.1次。Bar教授主要從事的研究包括RNAi，miRNAs以及small RNAs分子等方面。

轉(zhuǎn)錄組（transcriptom）廣義上指某一生理?xiàng)l件下，細(xì)胞內(nèi)所有轉(zhuǎn)錄產(chǎn)物的集合；狹義上指所有mRNA的集合。蛋白質(zhì)是行使細(xì)胞功能的主要承擔(dān)者，蛋白質(zhì)組是細(xì)胞功能和狀態(tài)的zui直接描述，而由于目前蛋白質(zhì)實(shí)驗(yàn)技術(shù)的限制，轉(zhuǎn)錄組成為研究基因表達(dá)的主要手段。轉(zhuǎn)錄組是連接基因組遺傳信息與生物功能的蛋白質(zhì)組的必然紐帶，轉(zhuǎn)錄水平的調(diào)控是目前研究zui多的，也是生物體zui重要的調(diào)控方式。轉(zhuǎn)錄組測(cè)序可以得到特定條件下所有mRNA轉(zhuǎn)錄本的豐度信息，從而發(fā)現(xiàn)新的轉(zhuǎn)錄本和可變剪接體。

以往的轉(zhuǎn)錄組測(cè)序原理是當(dāng)基因轉(zhuǎn)錄成RNA時(shí)，聚合酶延伸到超過蛋白編碼的部分，來形成“3'未翻譯區(qū)域” （UTR），該區(qū)域含調(diào)控性序列并幫助翻譯。腺嘌呤被添加到3'UTR，轉(zhuǎn)錄組測(cè)序的標(biāo)準(zhǔn)方法基于 “poly（A）”尾巴的結(jié)合。

而在這篇文章中，研究人員提出了一種3P-Seq（poly（A）-position profiling by sequencing）方法，并利用這種方法分析線蟲的3' UTR，獲得了8580個(gè)額外的UTRs，從而解析了線蟲3' UTR的形成，調(diào)控和進(jìn)化。

這種3P-Seq的原理如下圖，首先將一種配體（splint-ligation）與帶有*引物綁定位點(diǎn)的poly（A）結(jié)合（步驟1），然后通過T1核酶部分消化（步驟2）， 再洗脫捕捉poly（A），并且與加入的dTTP作用。之后再利用RNase H釋放，zui終用到高通量測(cè)序中去。這種方法精度更高，而且可以避免“poly（A）”方法的局限性。

Bar教授研究組近期還發(fā)現(xiàn)了MiRNA影響mRNA的機(jī)制，他們發(fā)現(xiàn)miRNA主要通過使目標(biāo)mRNA失去穩(wěn)定性來發(fā)揮作用，而不是通過抑制它們的翻譯來發(fā)揮作用。這與之前的研究發(fā)現(xiàn)并不相同。

原文摘要：Formation, regulation and evolution of Caenorhabditis elegans 3′UTRs

Post-transcriptional gene regulation frequently occurs through elements in mRNA 3′ untranslated regions （UTRs）1, 2. Although crucial roles for 3′UTR-mediated gene regulation have been found in Caenorhabditis elegans3, 4, 5, most C. elegans genes have lacked annotated 3′UTRs6, 7. Here we describe a high-throughput method for reliable identification of polyadenylated RNA termini, and we apply this method, called poly（A）-position profiling by sequencing （3P-Seq）, to determine C. elegans 3′UTRs. Compared to standard methods also recently applied to C. elegans UTRs8, 3P-Seq identified 8,580 additional UTRs while excluding thousands of shorter UTR isoforms that do not seem to be authentic. Analysis of this expanded and corrected data set suggested that the high A/U content of C. elegans 3′UTRs facilitated genome compaction, because the elements specifying cleavage and polyadenylation, which are A/U rich, can more readily emerge in A/U-rich regions. Indeed, 30% of the protein-coding genes have mRNAs with alternative, partially overlapping end regions that generate another 10,480 cleavage and polyadenylation sites that had gone largely unnoticed and represent potential evolutionary intermediates of progressive UTR shortening. Moreover, a third of the convergently transcribed genes use palindromic arrangements of bidirectional elements to specify UTRs with convergent overlap, which also contributes to genome compaction by eliminating regions between genes. Although nematode 3′UTRs have median length only one-sixth that of mammalian 3′UTRs, they have twice the density of conserved microRNA sites, in part because additional types of seed-complementary sites are preferentially conserved. These findings reveal the influence of cleavage and polyadenylation on the evolution of genome architecture and provide resources for studying post-transcriptional gene regulation.

來源：生物通