Identification and characterisation of novel piRNA processing factors in C. elegans

优雅线虫中新的piRNA加工因子的鉴定和特性。小的RNA分子可以在基因表达途径中发挥调节作用。目标的精确识别是基于碱基配对的，因此sRNA作为特异性因子，因此，它们的处理需要严格控制。其中一个保守的小RNA沉默途径是所谓的Piwi途径。这一途径是生殖细胞中控制转座子的主要监测系统之一，由称为piRNAs的小RNA控制。鉴于转座子序列的广泛多样性和多变性，需要很好地控制piRNA序列谱系，以避免宿主序列的识别。这是通过选择特定的转录本作为piRNA前体，然后进行不同的处理步骤来实现的。一般来说，对选择过程和加工步骤都不太了解。在线虫中，piRNAs来自特定的短RNA聚合酶II转录本。在此之后，它们被一种名为PETISCO的专用蛋白质复合体结合，这种复合体允许通过一种尚未确定的核酸酶来处理它们的5‘端。目前尚不清楚这一步骤是不同活性的汇编，如去帽，随后的外切核糖核酸酶，还是由不同的内切核酸酶驱动的。我们提供的初步数据暗示，内切核糖核酸酶是由一个异源二聚体蛋白复合体组成的，该复合体特异性地作用于线虫中与PETISCO结合的piRNA前体。拟议的工作计划旨在巩固这一假设，并了解这种新的酶活性。我们想了解它的三维结构，以及它是如何与PETISCO联系在一起的。此外，我们还鉴定了另外两个在piRNA生物发生中发挥作用的因子，它们在结构域水平上类似于构成二聚体核酸内切酶的蛋白质。我们的数据表明，基于piRNA加工核酸酶的两个亚基之一，可能存在其他类型的核酸酶的潜在模块化异二聚体组装。我们的目标是从生化和组织水平上了解这些复合体的功能(S)。

Identification and characterization of novel piRNA processing factors in C. elegans.Small RNA molecules can act as regulators in gene expression pathways. The precise recognition of targets is based on base pairing, and the sRNA thus acts as specificity factors, and consequently, their processing needs to be tightly controlled. One of the well-conserved small RNA silencing pathways is the so-called Piwi pathway. This pathway is one of the main surveillance systems in germ cells to keep transposable elements under control and is steered by small RNAs known as piRNAs. Given the wide diversity and varying nature of the transposon sequences, the piRNA sequence repertoire needs to be well controlled to avoid the recognition of host sequences. This happens by selecting specific transcripts as piRNA precursors, followed by distinct processing steps. Neither the selection process nor the processing steps are in general well understood.In the nematode Caenorhabditis elegans (C. elegans), the piRNAs are derived from specific, short RNA polymerase II transcripts. Following this, they are bound by a dedicated protein complex named PETISCO, which allows the processing of their 5′ end by an as-yet-unidentified nuclease. Whether this step is a compilation of different activities, such as de-capping, followed by exoribonucleolytic trimming, or is driven by a distinct endoribonucleolytic enzyme is unknown.We present preliminary data that implicate that the endoribonuclease is constituted by a heterodimeric protein complex that specifically acts on PETISCO-bound piRNA precursors in C. elegans. The proposed work plans are aimed to solidify this hypothesis and to understand this novel enzymatic activity. We want to learn about its 3-dimensional structure and how it is connected to PETISCO. In addition, we identified two additional factors that play a role in piRNA biogenesis and resemble proteins constituting dimeric endonuclease on the domain level. Our data suggest a potential modular heterodimeric assembly of potentially other types of nucleases, based on one of the two subunits of the piRNA processing nuclease. We aim to understand the function(s) of those complexes both at the biochemical and organismic levels.