Identification, Expression And Function Of Small Nucleolar And MRNA-Like Non-Coding RNAs In Pigs

Besides conventional protein-coding genes, a new group of genes in the porcine genome were identified which function directly as structural, catalytic or regulatory RNAs. They were entitled the non-protein-coding RNA genes (ncRNA genes). Scientists had found that they were involved in a number of critical cellular events and focused in the genes increasingly in recent years. Considering the intensive research in microRNAs and piRNAs, we focused in the longer ncRNA groups, such as the small nucleolar RNAs (snoRNAs) and messenger RNA-like non-coding RNAs (mlncRNAs), instead. We combined the adaptor-ligation with the polyacrylamide gel electrophoresis (PAGE) protocols to isolate the target genes, characterized their primary structures and expression patterns and studied the possible function. The main protocols and results were as follows:1. Identification of new genes of porcine snoRNAs and mlncRNAsIn the research targeting snoRNAs, we combined the adapter-ligation and the PAGE, recovered the RNAs of typical length and constructed a cDNA library, representing the snoRNAs in skeletal muscle of 3 developmental periods from both Landrace and Tongcheng pigs. By sequencing and prediction in silico, we obtained 120 new porcine snoRNA genes in total. For isolating porcine mlncRNAs,512 pairs of cDNAs from an evolutionarily conserved full-length ncRNA library were used as seeds for expressed sequence tag (EST) hunting in pig. After assembly from EST,119 porcine mlncRNAs were obtained. Next, thresholds were set according to the reference and these contigs were screened one by one. At last,93 candidate porcine mlncRNAs were obtained by computational prediction and screening.2. Characterization primary structure of porcine snoRNAs and mlncRNAsAccording to the primary structure of the 65 sequenced snoRNA genes, we characterized their distributing of lengths and GC contents and checked the conservation of sequences of the C, D, H and ACA boxes one by one. The results indicated that the values of both lengths and GC contents of H/ACA box snoRNA showed smaller standard deviations but larger averages, which were opposite to those of C/D box snoRNAs. Meanwhile, the conservation of sequences of D and ACA boxes were higher than those of C and H boxes, respectively. Analysis of 8 representative candidates revealed that the mlncRNA genes shared low conservation among species. The sequences of these genes showed similarities only among the eutheria. 3. Mapping of porcine snoRNAs and mlncRNAsBased on public database,91 genes of 120 newly identified snoRNAs were mapped into porcine genome. Among them,22 genes were assigned to chromosome 9, while 4 chromosomes have not contained any genes found in this research. Referring to the genomic distribution of corresponding human homologous genes, we summarized the 91 porcine genes into 9 different genomic distribution patterns. Aligned with porcine genomic databases,72 of the 93 genes were mapped to the porcine genome. Among them,8 were assigned to chromosome 1, while none was found in chromosome 17 and X.4. Function studies of porcine snoRNAs and mlncRNAsNext, we deduced the guide sequences, which function in targeted RNAs modifying, in each porcine snoRNA from the alignment with human genes firstly. Then, we predicted the possible modified loci in porcine U6 snRNA,5.8S and 18S rRNA, according to the rule of "D/D’+5" in C/D box and the analogue in H/ACA box snoRNA respectively. Moreover, we found that majority of the loci were conserved evolutionally. One of the mlncRNA genes, sTF35495, was found to be precursor of a putative porcine microRNA, the microRNA-568, evidenced by its secondary structure and alignment with human and mouse corresponding pre-microRNA genes. By RACE PCR, we determined that the full length of sTF35495 was 3 kb. The protein-coding potential of this RNA was tested in silico with no significant finding.5. Expression patterns of porcine snoRNAs and mlncRNAsWe tested the expression patterns of 6 snoRNA genes in skeletal muscle of different developmental periods of Landrace and Tongcheng pigs. The results showed that the expression patterns of 6 genes were significantly different between 2 breeds in development. All expression levels of genes declined firstly and then rose in Tongcheng pigs while those merely rose in Landrace. Semi-quantitative RT-PCR analysis of 8 mlncRNA genes revealed two distinct expression profiles, in which some genes demonstrated obvious tissue-specificities while others were expressed ubiquitously in vivo. Furthermore, expression levels of two genes were further validated by real-time PCR.