Identication And Functional Verification Of LincRNA And SORF In Porcine Multiple Tissues

Long intergenic non-coding RNA constitute a part of the mammalian transcriptome, the length of them is longer than 200 bp and they do not have the coding capacity. It has reported that lincRNA function as enhancer to regulate gene expression. In addition, research results have indicated that some lincRNA play important roles in biological processes, such as imprinting, X-inactivation, and chromatin remodeling. Currently, thousands of lincRNA in the human genome and other species have been uncovered, but the reports about the annotation and function research of lincRNA in pig genome are rare.The main research results about lincRNA in porcine multiple tissues are as follows:(1) Filtering lincRNA from RNA-Seq data about porcine multiple tissues and characteristics of them: We downloaded the RNA-Seq data from NCBI, used TopHat and Cufflinks to map the reads to reference genome and align them to be transcripts. Referd to the characteristics of lincRNA which have been reported such as length, exon number, coding potential, and expression level, we finally identified 9982 transcripts in pig genome. The result of the sequence length distribution suggested: most novel identified lincRNA is shorter than protein-coding genes; most lincRNA have 2 exons, all of them generally have exon number between 2 and 4; the coding potential of novel identified lincRNA is lower than zero, and it is similar to 47 annotated lincRNA in Ensembl data base, lower than annotated protein-coding genes in Ensembl data base; the expression level is obviously lower than annotated protein-coding genes.(2) Validation of novel identified lincRNA in transcript level by RT-PCR and determination of 3’ends of them: We viewed lincRNA by IGV and validated 27 transcripts by reverse-transcribe PCR with 8 different tissues(heart, liver, spleen, lung, kidney, longissimus dorsi, subcutaneous fat, brain) of Tongcheng pigs; 26 transcripts were amplified successly and had products. Then we chose 20 transcripts of them on the basis of agarose gel electrophoresis results of RT-PCR to validate 3’ends of them by 3’ rapid amplification of cDNA ends. Finally we successly validated 3’ends of 6 transcripts, one of them can encode micropeptide RPL41.(3) Multiple sequence alignment of RPL41 among species: The result showed that more than 90% of RPL41 nucleic acid sequence identified to sequence of human, mouse, rat, while protein RPL41 which is encoded by ORF of 75 bp completely identified to amino acid sequence of RPL41 from other species. Looking at DNA sequence in the pig genome, we discover annotation of the gene is not complete.There exists a gap region after the exon 3, the sequence of exon 4 has been ascertained by 3’RACE. PCR result showed that DNA sequence of intron 3 of RPL41 has been amplified successfully.(4) Influence on luciferase of RPL41 by reporter experiments: Result displayed that RPL41 did not function like an enhancer, but notably inhibit the expression of luciferase, it is different with some lncRNA with have enhancer-like function.(5) Construction of eukaryotic expression recombinant plasmid pcDNA3.1-RPL41-EGFP and expression of fusion protein: We can see green light of fusion protein with the fluorescence microscope and we also proved the expression of fusion protein by Western Blot. The results suggested that RPL41 can encode micropeptide.(6) Analysis of differentially expressed genes after overexpression and interference of RPL41: The data of researching porcine domestication which is downloaded from SRA database displays there are 3421 differentially expressed genes in total and the expression of RPL41 is notably higher in domesticated pig than in wild boar. The data of next generation sequencing after overexpression and interference of RPL41 in PK-15 cells shows there are 618 differentially expressed genes, GO analysis indicates some of them are related to transcription. Analysis shows that there are 60 differentially expressed genes appear in two sets of data. We selected 3 genes to test the expression by Q-PCR, the result is almostly the same as result of RNA-Seq.We established a bioinformatics procedure of filtering lincRNA about porcine multiple tissues RNA-Seq data, analysised the characteristics of novel identified lincRNA and summarized them. Expression characteristics of them have been initially validated by experiments of RT-PCR and 3’RACE. We identified a lincRNA which actually can encode a micropeptide and initially analysis its function by GO analysis and experiments. Research results can enrich function annotation of pig genome and provide a reference for future identification and function research of micropeptide genes.