Identification and Characterization of Novel Somatic and Germ Cell Small RNA Species

Abstract

Small non-protein-coding RNAs are essential, ubiquitous molecules found in every cell. Their functions range from post-transcriptional regulation of mRNA expression to epigenetic control of the genome. Small noncoding RNAs also have crucial functions in the ribosome and spliceosome. Block of small RNA biogenesis and inactivation of effector complex proteins lead to embryonic lethality and loss of cell function. Because of their necessity in biological processes, they are of great interest in current research. Although small RNA research is in full throttle, many ambiguities and obstacles in this field still need to be addressed. This work resolves some of these roadblocks and expands our knowledge of existing small RNAs and their expression in somatic and germ cells. To properly characterize the function of small RNAs in a cell, it is essential to define the small RNA transcriptome. This can be accomplished through next-generation sequencing of small RNA. Unfortunately, current work in small RNA tends to concentrate on only one type of small RNA, predominantly miRNA, due to their already defined function, biogenesis, and structure. In addition, next-generation sequencing data generate millions of reads, and it is complicated to consider the entire small RNA transcriptome, as no comprehensive annotation pipeline is currently available. To further this field, we set out to define a small RNA annotation pipeline. To aid in this effort, we developed a graphic user interface program for small RNA data processing and annotation. We validated this pipeline by defining the Sertoli small RNA transcriptome with next-generation sequencing data. Sertoli cells represent the predominant somatic cell type within the testis and are considered "nurse cells" to developing male germ cells. Male gametes, sperm, are produced through spermatogenesis in the testis. Sertoli cells are in direct contact with all developing germ cells during spermatogenesis and normal Sertoli cell functions are required for normal germ cell development. Small RNAs help regulate dynamic transcriptomes, and we hypothesized that the Sertoli small RNA transcriptome needs to be dynamic due to the different germ cell stages Sertoli cells support. Indeed, all small RNA classes were found within the Sertoli cell and many novel RNAs were also identified in this study. Altogether, we successfully defined the entire small RNA transcriptome of Sertoli cells and developed a small RNA annotation pipeline for the comprehensive annotation of known and novel small RNAs. In our research of small RNAs, we identified a class of somatic small RNAs approximately 30 nucleotides long. In the male gonad a similar population has been defined as PIWI-interacting RNAs or piRNAs. piRNAs are approximately 30 nucleotides long and associate with PIWI proteins. Because PIWI proteins are germ cell exclusive, the somatic counterparts discovered cannot be considered piRNAs. Instead we termed them piRNA-like or pilRNA for their similarities to piRNAs. Our aims of this research were to compare pilRNAs to germ cell piRNAs, to determine if pilRNAs require known small RNA biogenesis proteins, and to resolve potential pilRNA functions in somatic cells. Further analysis of these pilRNA species in somatic cells revealed that some pilRNA sequences are identical to piRNAs expressed in the testis. In addition, pilRNAs are found expressed from the same genomic clusters as germ cell piRNAs. These somatic small RNAs seem to mimic pachytene piRNAs, the second wave of piRNAs produced through spermatogenesis, and do not perform ping-pong self-amplification, but do not require the same machinery for production. pilRNAs also do not require other small RNA (miRNA and endo-siRNA) producing proteins such as DICER and DROSHA indicative of these somatic sequences being a novel class of small RNAs. Furthermore, we propose that pilRNAs may have a post-transcriptional function as pilRNAs are partial complementary to mRNA transcripts at the 3' untranslated regions. In conclusion, germ cell piRNAs have a novel somatic counterpart, pilRNAs, which are found abundantly expressed in somatic cells and tissues.In the germ cell RNA field, for many years researchers solely concentrated on sperm as paternal DNA deliverers to the oocyte. It was thought that since sperm are transcriptionally and translationally inactive they would not require RNA contents. It has been ten years since the first discovery of sperm mRNA and the dissolution of the idea of sperm functioning through DNA delivery. To date sperm RNA studies have been plagued with inconsistencies. These inconsistencies stem from improper RNA isolation procedures and the difficulty of lysing disulfide bond ridden sperm. This work resolves these inconsistencies by providing a RNA isolation method that gives consistently reproducible results. Using this RNA isolation procedure, we were able to define the small RNA transcriptome of human and mouse whole sperm and sperm head. Differential analysis of sperm head compared to whole sperm samples revealed that whole sperm showed an increase in mature miRNAs in comparison to head miRNA expression indicative of increased RNA interference being performed in the tail, cytoplasm, and outer membrane. In contrast, sperm head have elevated expression in RNAs fragmented from larger small RNAs such as tRNA-derived and rRNA-derived small RNAs. Particularly, the tRNA-derived population of small RNAs is highly enriched in sperm nucleus. Based on genomic location comparisons, tRNA-derived small RNAs are predominantly generated from regions overlapping gene-coding boundaries in the genome. Because tRNA-derived fragments are the predominant species in the sperm head and their genomic loci coincide with gene loci, we predict that tRNA-derived small RNAs hold epigenetic roles in sperm.In summary, this work expands our current knowledge of small RNAs, as well as removes some previously hindering roadblocks that prevented advancements in small RNA research. Developing a small RNA annotation pipeline for the facile categorization of known and novel species of small RNA has limitless applications for small RNA research. We also identified and characterized a novel population of somatic small RNAs similar to piRNAs. In addition, we were able to develop a protocol for sperm RNA isolation that yielded consistent RNA profiles and defined mouse and human sperm transcriptomes using this protocol. These discoveries not only contribute to the small RNA research currently available, but provide other researchers with tools to expand their research.