| 98% of the human genome is pervasively transcribed to non-coding transcripts,the so called “RNA dark matter”.Such transcripts are classified into 2 categories,the long(> 200 nt)and small(< 200 nt)noncoding RNAs,based on their length.The human small noncoding RNAs(snc RNA)are quite abundant and diverse.They consist of well-characterized structural snc RNAs,such as ribosomal(r),transfer(t),and also regulatory snc RNAs with well-understood functions and mechanisms of action,including small interfering(si),micro(mi),and PIWI-interacting(pi).However,the functions,mechanisms of action,and biological relevance of the vast majority of snc RNAs remain unknown.Since several known classes of snc RNAs have been found playing crucial roles in multiple biological processes,it is assumed that many unannotated snc RNAs are also involved in important biological functions.Nevertheless,direct evidence of functionality is absent for most snc RNAs,especially for snc RNAs that are often considered as stable degradation products of longer RNAs.In this study,we aimed to functionally characterize human snc RNAs by studying two questions:(1)whether the unannotated snc RNAs have biological function and(2)whether a significant fraction of unannotated snc RNAs belongs to a known class of snc RNAs,specifically,mi RNA.Therefore,in this study,we,for the first time,developed and applied a novel high-throughput phenotyping approach to study the biological functions of unannotated snc RNAs.In this approach,we first constructed a screening system for unannotated snc RNAs,and thus a significant number of unannotated snc RNAs located in the functional regions of the genome were identified based on the small RNA sequencing data of the K562 cell line.Then,a stable inducible over-expression system was constructed in human cells for the identified unannotated snc RNAs,and was used to study the effects of snc RNAs on cell growth and survival.Finally,by high-throughput sequencing approach,functional unannotated snc RNAs that can promote or inhibit cell growth and survival were screened out.The results revealed that the overexpression of 43.1% of the newly identified unannotated snc RNAs could inhibit cell growth and survival,indicating that these snc RNAs have potential biological functions.In order to further explore the types of newly discovered functional unannotated snc RNAs,we developed a high-throughput screening approach to identify novel functional snc RNAs — mi RNAs at the transcriptome-wide level.This approach is based on the biogenesis pathway and sequence structural features of mi RNAs.In the biogenesis of most mi RNAs,a precursor mi RNA with a hairpin structure was first produced by Drosha cleavage,which is further processed to guide-strand and complement passenger-strand mi RNAs with length of 20–25 nt.In the approach,a Drosha stable silencing system was construct,and Drosha silencing relevant snc RNAs were thus identified.Then,based on the sequence conservation and structural characteristics of Drosha-dependent mi RNAs,a model built and used to screen Drosha silencing relevant snc RNAs for potential novel mi RNAs.Finally,an algorithm model was developed based on the hairpin structural features of precursor mi RNAs,and used to predict the complementary strand of the potential novel mi RNAs.The results were then compared with all human small RNA sequencing databases to prove the biogenesis of novel mi RNAs in vivo.By this approach,2245 novel mi RNAs were discovered in the K562 transcriptome in this study.Overall,in this study,we developed a novel high-throughput approach to study the function of snc RNAs,and discovered that more than 40% of unannotated snc RNAs appear to have potential biological function.Furthermore,we found at least 2245 new mi RNAs in one human cell line.These results suggest that mammalian snc RNAs transcriptome harbors multitude of unknown snc RNAs,whose structures and functions need to be further studied.The high-throughput approaches established in this study will facilitate the further study of snc RNAs. |
