Identification And Expression Analysis Of 50-500nt Ncrnas From The Genome Of Domesticated Silkworm Bombyx MORI

It is becoming clear that a large number of genomic transcripts in both prokaryotes and eukaryotes are non-protein coding RNAs (ncRNAs). Small ncRNAs, such as miRNAs, siRNAs and piRNAs have been widely studied, and accumulating evidences show that they play important and diverse roles in gene regulation. Moreover, snRNAs and snoRNAs have been showed to be closely related to development and human disease. It is necessary to identify novel ncRNAs in different organisms and probe the roles which they may play in development.Bombyx mori is the model insect of the order Lepidoptera, with considerable economic importance. The B. mori genome was completely sequenced in 2004 with the size of～432 Mb. Although ncRNAs have been studied extensively in D. melanogaster, less work has been carried out in B. mori. Recently,420 novel miRNA candidates were found in silkworm,248 of which have either egg- or pupa-specific expression. Given the large number of ncRNAs existing in Drosophila, we predicted that there might be many ncRNAs in B. mori as well. The main purpose of the research is to discover novel ncRNAs in the silkworm genome as many as possible and to identify the function of the novel ncRNAs. With an experimental RNomics, we constructed the cDNA library of 50-500nt ncRNAs in B. mori. The main results were as following.In our library,194 candidates for novel, small non-messenger RNAs were identified, which contained 45 C/D box snoRNAs,83 H/ACA box snoRNAs, and 58 transcripts lack of conserved motifs or secondary structure hallmarks. In addition, we also found 2 novel C/D-H/ACA box "hybrid"snoRNAs and one novel SRP RNA.By comparing our novel ncRNAs with the known ones in other organisms, we found that except the highly conserved snRNAs and SRP (both with the identity above 86.3%), the remaining ncRNAs were not conserved or exhibited very low sequence similarity to other ncRNAs. Analyzing for antisense elements and characteristic secondary structures of snoRNAs, we found that 8 C/D box snoRNAs and 36 H/ACA box snoRNAs were conserved among the novel transcripts. However, the other 141 ncRNAs couldn’t find any homologue, which were silkworm-specific.The genomic organization of ncRNAs was various in the silkworm.103 ncRNAs were located in the introns of protein coding genes,80 were transcribed from the intergenic regions, four ncRNAs overlapped with the first or last exons of coding genes, another four were complementary to the introns of coding genes, and three ncRNAs were origined from the predicted transposon regions. Further analysis indicated that the intron-origin ncRNAs in our library followed the pattern of one-ncRNA-per-intron, which was very different to that in Drosophila, where C/D box snoRNA had the pattern of one-snoRNA-per-intron, but the H/ACA box snoRNAs were transcribed from the genome with a pattern of cluster. In our library, besides two intergenic ncRNA gene clusters, the others were all existed separately, which was also very different to that in Drosophila.With targets predicted by snoScan and snoGPS, we found 22 C/D box snoRNAs could guide the 2’O-methylation at 27 rRNA, snRNA and tRNA sites, and 49 H/ACA box snoRNAs direct to 72 pseudouridylation sites of rRNAs, among which,3 C/D box snoRNAs were found to guide the 2’-O methylation of four tRNAs. Two C/D-H/ACA box ’hybrid’snoRNAs were predicted to guide three pseudouridylation sites in 18S rRNAs and 5.8S rRNAs. Another three were predicted to target 3 mRNAs.In order to elucidate the expression patterns of these novel ncRNAs, microarray and Northern blot were performed,132 probes were successfully designed and spotted on the slides, results showed that 36 of which had significantly differential expression during development, and most of them had relatively more expression in egg and adult stages. The expression pattern of these novel ncRNAs during a specific developmental stages such as larva and pupa were further analyzed, results showed that the expression of the selected ncRNAs showed only minor difference over the course of larval development, with exception of Bm-86 having more expression in the fifth instar larva. However, the expression of Bm-51, Bm-86, Bm-152 and Bm-160 decreased during pupation.Some intron-origin ncRNAs had consistent expression pattern with their host genes, which indicated that they may transcribed from splicing dependant manner and participate in the similar cellular process. The intergenic-origin ncRNAs had various relationships with their up-or-down stream genes, and the ncRNAs transcribed from the sense-antisense gene pairs can negatively or positively participate in the regulation of their "sense" genes. All of which indicated that the regulator roles of ncRNAs was intricate.In addition, we found some ncRNAs hybrided two bands in Northern blot results, which indicated that they might transcribed with an alternative splicing manner.Our results provided the first genome-wide survey of genomic organization, biogenesis and developmental expression of ncRNAs with length of 50-500 nt in B. mori, which will provide a realible theoretical basis for further researches on silkworm.