Genomic Scale Studies Of NcRNA Composition Of The Mouse Mitotic Chromosomes

Mitotic chromosomes are one of the most important and commonly recognized sub-cellular structures in eukaryotic cells.It is a highly complex advanced structure which is composed of DNA,protein and RNA's ordered folding.Nevertheless,due to various limitations of the technology and theory,the basic information necessary to understand their structure and assembly,such as their composition,is still lacking.Sub cellular profiling should be an important initial step to study mitotic chromosome related molecular mechanisms and enrichment of specific molecular species in well characterized cellular structures will provide a basis not only for correlative examinations but also mechanistic studies.In recent years,the rapid development of omics technology has made the study of metaphase chromosomes and once again become a hot frontier.Recent proteomic studies have begun to fill this void,identifying hundreds of RNA-binding proteins bound to mitotic chromosomes.However,by contrast,there are only two RNA species(U3 snRNA and rRNA)that are known to be associated with the mitotic chromosome,suggesting that there are many mitotic chromosome-associated RNAs(mCARs)not yet identified.To identify the RNA composition on mitotic chromosomes,I first optimized a procedure employing sucrose gradient centrifugation to isolate mitotic chromosomes with a minimal amount of loosely bound(perhaps cytoplasmic)RNA or ribonuclear proteins by salt treatment.This concentration of salt was identified as the highest concentration at which neither the protein composition associated with the mitotic chromosome(in particular the histone H1)nor the chromosome morphology are significantly altered from that in the absence of the incubation with the high salt buffer.Then,using a targeted protocol based on 5'-tag sequencing to profile the mammalian mCAR population,we first report the identification of 1279 mCARs,the majority of which are ncRNAs,including lncRNAs and snoRNAs that exhibit greater conservation across 60 vertebrate species than the entire population of lncRNAs and snoRNAs.There is also a significant enrichment of specific SINE RNAs.Our data also confirmed the existence of 500 ncRNA that have not yet been annotated.RNA FISH and other technology validation results are consistent with the high throughput sequencing results.On the other hand,by using different library construction method,this study analyzes the composition of the metaphase chromosomal small RNAs by high-throughput sequencing and identified 8441 small mCARs including 301 annotated miRNAs.Overall,the mCARs identified here,together with the previous proteomic and genomic data,constitute the first comprehensive catalogue of the molecular composition of the eukaryotic mitotic chromosomes.And this study provides an important basis to further explore structure and molecular mechanisms of the mitotic metaphase chromosome.