Bacterial group II introns: splicing, function and evolution

我的研究计划的长期目标是解决与II组内含子的剪接，功能和进化相关的基本问题。我们的模型系统是来自工业上重要的革兰氏阳性细菌乳酸乳球菌的Ll.LtrB II组内含子。II组内含子是从前mRNA自我剪接的大核酶。真核细胞核内含子和片段化的II组内含子使用完全相同的剪接途径，强烈表明II组内含子和核内含子具有共同的进化起源。第二组内含子的另一个迷人的特征是，在它们的自催化切除之后，它们有可能通过逆转录归巢侵入新的相同DNA位点，并通过逆转录转座侵入新的非同源DNA位点。目标1：我们表明，Ll.LtrB组II内含子可以在多个位置被片段化成两个或三个片段，并且仍然组装和剪接。这项工作强烈支持进化理论，声称组II内含子是真核细胞核内含子的祖先，并且五个小的核RNA是剪接体机制的一部分，来自组II内含子片段。我们拟通过研究内含子片段在体内的折叠和组装过程，以及内含子编码的蛋白LtrA在其折叠和组装过程中的作用（1.1），来鉴定和研究L.乳酸菌（1.2），并评估Ll.LtrB是否可以进一步片段化成四个和五个片段，并且仍然组装和反式剪接（1.3）。目标二：我们最近发现，很大比例的Ll.LtrB是以完美的RNA环形式释放的，这表明该内含子不仅在L.乳酸。总体而言，我们的工作表明，环化是一个保守的第二组内含子剪接途径产生剪接产物具有潜在的新的有趣的生物学功能。我们建议完成主要II组内含子环化途径的表征（2.1），以进一步详细研究新的Ll.LtrB环化途径（2.2）及其剪接产物的功能：内含子RNA环（2.3）和嵌合mRNA（2.4）。目标3：我们提供了第一个实验证明了公认的和长期存在的理论，即第二组内含子可以在不同物种之间横向转移。我们的工作有力地表明，与其他移动的元件相关的II组内含子的意外高代表性反映了这些内含子在细菌细胞之间穿梭所具有的进化优势。我们提出研究通过细菌界中的接合的Ll.LtrB侧向转移的宿主范围（3.1），以评估Ll.LtrB在细菌生物膜内的传播潜力（3.2），以研究通过噬菌体转导（3.3）和通过细菌膜囊泡（3.4）的Ll.LtrB侧向转移。

The over-arching and long-term goal of my Research Program is to address fundamental questions related to the splicing, function and evolution of group II introns. Our model system is the Ll.LtrB group II intron from the industrially important Gram-positive bacterium Lactococcus lactis.******Group II introns are large ribozymes that self-splice from pre-mRNAs. Eukaryotic nuclear introns and fragmented group II introns use the exact same splicing pathway strongly suggesting that group II and nuclear introns have a common evolutionary origin. Another fascinating characteristic about group II introns is their potential, following their autocatalytic excision, to invade new identical DNA sites by retrohoming and non-homologous DNA sites by retrotransposition.******Aim 1: We demonstrated that the Ll.LtrB group II intron can be fragmented in two or three pieces at multiple locations and still assemble and splice in trans. This work strongly supports the evolutionary theories claiming that group II introns are the progenitors of eukaryotic nuclear introns and that the five small nuclear RNAs, that are part of the spliceosome machinery, were derived from group II intron fragments. We propose to study how the pieces of fragmented introns fold and assemble in vivo and the contribution of the intron-encoded protein LtrA in their folding and assembly process (1.1), to identify and study Ll.LtrB trans-splicing factors in L. lactis (1.2) and assess if Ll.LtrB can be further fragmented in four and five pieces and still assemble and trans-splice (1.3).******Aim 2: We recently showed that a significant proportion of Ll.LtrB is released as perfect RNA circles demonstrating that this intron is not only released as lariats in L. lactis. Overall, our work shows that circularization is a conserved group II intron splicing pathway generating spliced products with potentially new interesting biological functions. We propose to complete the characterization of the primary group II intron circularization pathway (2.1), to study in further details the new Ll.LtrB circularization pathway (2.2) and the function of its splicing products: the intron RNA circles (2.3) and the chimeric mRNAs (2.4).******Aim 3: We provided the first experimental demonstrations of the well-accepted and long-standing theory stating that group II introns can be laterally transferred between diverged species. Our work strongly suggests that the unexpectedly high representation of group II introns associated with other mobile elements reflects the evolutionary advantage held by these introns shuttling between bacterial cells. We propose to study the host range of Ll.LtrB lateral transfer by conjugation in the bacterial kingdom (3.1), to evaluate the dissemination potential of Ll.LtrB within bacterial biofilms (3.2), to investigate the lateral transfer of Ll.LtrB by bacteriophage transduction (3.3) and through bacterial membrane vesicles (3.4).