Transcription And Promoter Analysis Of Drosophila Intronic MicroRNA-281-1/2 Located In Alternative Spliced Region

MicroRNA(miRNA) is an abundant class of small(～22 nucleotides),endogenous noncoding RNAs that function at post-transcriptional gene regulation.Some miRNAs are located in the intergenic region,whereas other miRNAs are intronic.Until now,little has been known about the transcription of intronic miRNA and its relationship with host gene. We found that some intronic miRNAs are located in the alternative regions of host genes. For example,Drosophila miR-281 cluster is in either the first intron or the 5'UTR of the ODA gene.Here,the transcription of miR-281 and its relationship with the host gene were studied.The promoter of miR-281 was also analyzed.This work is helpful for better understanding the complex and fine-tuning regulation of miRNA transcription.1.Drosophila miR-281 transcription analysisThe precursor of miR-28 lcluster was successfully detected by RT-PCR.The full length of the miR-281 transcripts was amplified with the RACE technique.According to RACE results,the transcripts of miR-281 were produced by three different transcription start sites, named as TSS1,TSS2 and TSS3.A TATA box is found at the 350 bp upstream of TSS1.All detected transcripts were polyadenylated,with a canonical polyadenylation signal (AATAAA) near 3' terminus.The conservation of genomic regions flanking three TSSs are higher than average levels.Though miR-281 is located in the intron of ODA gene,it is independently transcribed with its own promoter.2.Secondary structure analysis,target predicted and evolution analysis of miR-281Dme-miR-281 gene that includes two precursors and four mature miRNA belongs to mir-46 gene family.Only one nucleotide variance exists at the arm regions of pre-miR-281-1 and pre-miR-281-2,whereas up to 13 nucleotide changes appear in the loop region.In the miRBase,there are seven homolog precurors(corresponding to eight homolog mature miRNA) of dme-miR-281.RNAFold and Mfold were used to predict the secondary structure of pre-miR-281 and pri-miR-281.In the structures of all three transcripts,pre-miRNA regions fold into typical hairpin structures.Eight-three protein coding genes were predicted to be the target of miR-281-1,and 69 mRNAs were targeted by miR-281-2.Among two groups of mRNA targets,61 mRNAs were targeted by both miR-281-1 and miR-281-2.The homologs of miR-281 are found in sixteen species from the UCSC database. Interestingly,the duplication of pre-miR-281 only appears in Drosophila melanogaster and its close species.In the miR-46 family tree inferred by the MrBayes algorithm,miR-281-1 and miR-281-2 are clustered into two different clades.3.Expression profile analysis and the relationship with its host geneExpression analysis showed that isoform RA,RB and RC of ODA gene expressed at every stage,whereas the transcripts of pri-miRNA281 were only detected in the third instar, pupa and adult.Real-time PCR showed that the abundance of isoform RA and RC is higher than that of isoform RB and miR-281.This is consistent with EST analysis results.There are more ESTs supporting isoforms RA and RC than RB and miR-281.The expression of isoform RA and RC varied at different development stages.However,there was no significant difference of the miR-281 among the different stages.These results suggested that alternative transcription start sites of the host ODA genes have little impact on the transcription of intronic miR-281 cluster.4.The promoter analysis of miR-281 and ChIP analysis of MycThe genomic reigons -1020 bp to +412 bp flanking the TSS were defined as putative promoter.By searching the TRANSFAC database,some well-known transcription factors such as Myc,C/EBP and CAD are predicted to bind to putative the promoter region of dme-miR-281.The binding sites are highly conserved between diverse organisms by Blaring the UCSC database.The genomic DNA binding with Myc protein was immunicipated by Chromatin Immunoprecipitation(CHIP) technique.ChlP-PCR with miR-281 primers confirmed the physical presence of Myc at the promoter region of miR-281.This suggested that Myc might participate in the transcription of miR-281 and provided a piece of evidence of independently transcription of intronic miR-281.The putative promoter ranging from -1020 bp and +412 bp flanking the TSS1 were amplified and ligated with pAC5.1 vector that contains green Fluorescent protein(GFP) as the report gene.The constructs were transected into the Drosophila S2.However,the fluorence of GFP protein could not be detected,indicating that the putative miR-281 promoter did not drive the expression of downstream GFE One possible reason is that miR-281 promoter has a low activity in Drosophila S2 cell as miR-281 only expresses at the third instar,pupa and adult stage.5.Efficiency analysis of multiple transcription start sites of miR-281To estimate the transcriptional efficiency of different TSSs,the real-time PCRs were carried out using isoform-specific primers.Two sets of cDNA template were produced.One is reversed transcribed using random hexamers as anchor primer.Another is reverse transcribed with Oligo(dT) primer.The results manifested that TSS3 might be a genuine TSS.Relative to TSS1 transcripts,TSS2 transcripts is 0.97 fold in the third instar and 0.83 fold at adult,suggesting that TSS1and TSS2 have similar transcriptional efficiency.The transcriptional efficiency of TSS3 is changable at different development stages.Generally, it has a higher efficiency than TSS1 and TSS3.