7 replies to this topic

[j123]

I am pretty new to this field. Does anybody know how to find the promoter region of an intronic miRNA? How to distinguish the intronic miRNA promoter with its host gene promoter? Many thanks.

[Jon Moulton]

I am pretty new to this field. Does anybody know how to find the promoter region of an intronic miRNA? How to distinguish the intronic miRNA promoter with its host gene promoter? Many thanks.

I don't think the intronic miRNA has an independent promoter, but rather that the host gene promoter causes transcription of the pre-mRNA containing the intronic miRNA and then the intronic miRNA is cleaved from the intron by Drosha. I am curious whether this is thought to happen after splicing, so that the miRNA is cleaved from the intron lariat. I don't see how it could happen prior to lariat formation without disrupting the pre-mRNA by cleaving the pre-mRNA into two pieces.

Please chime in!

[j123]

Thanks for your reply. One paper says that 26% of intragenic miRNAs may be transcribed from their own unique promoters. I used Tet-on Advanced system to express the whole host gene, and checked the miRNA expession by RT-PCR. I didn't see the increase of miRNA although the host gene expression dramastically increased. Thus, I think the miRNA I am interested in and the host gene come from different transcripts. But I am not sure if they share the same promoter. Any suggestions? Thanks again for your kind discussion.

I am pretty new to this field. Does anybody know how to find the promoter region of an intronic miRNA? How to distinguish the intronic miRNA promoter with its host gene promoter? Many thanks.

I don't think the intronic miRNA has an independent promoter, but rather that the host gene promoter causes transcription of the pre-mRNA containing the intronic miRNA and then the intronic miRNA is cleaved from the intron by Drosha. I am curious whether this is thought to happen after splicing, so that the miRNA is cleaved from the intron lariat. I don't see how it could happen prior to lariat formation without disrupting the pre-mRNA by cleaving the pre-mRNA into two pieces.

Please chime in!

[miRNA man]

Intergenic is different to intronic. But anyway, have you checked expression of any endogenous miRNA in your cell line? Some cancers and perhaps cancer cell lines have mutations in the miRNA biogenesis machinery.

As to Jon's questions, perhaps the Drosha processing isn't very efficient and so not all of the transcript is processed to pre-miRNA, leaving some of the host mRNA intact? I suppose though that if the lariat is long enough and has the correct secondary structure and/or Drosha recognition sequence then it could be precessed. As far as I know (and please correct me) the recognition sequence for Drosha hasn't been determined? If we knew that, it might be a lot easier to understand!

[Jon Moulton]

As to Jon's questions, perhaps the Drosha processing isn't very efficient and so not all of the transcript is processed to pre-miRNA, leaving some of the host mRNA intact? I suppose though that if the lariat is long enough and has the correct secondary structure and/or Drosha recognition sequence then it could be precessed. As far as I know (and please correct me) the recognition sequence for Drosha hasn't been determined? If we knew that, it might be a lot easier to understand!

I've read speculation that Drosha recognizes target based on secondary structure, not sequence. I'm not convinced either way.

If Drosha cuts after splicing occurs, the production of mature coding transcript could continue very efficiently. I like your point that only a fraction of transcript might be recognized by Drosha and thus pre-splicing excision of the stem-loop might not seriously impact expression of the host gene. We'll just toss that question onto the "biologist job security" pile .

[j123]

Thanks for the response. I mean intragenic, not intergenic. It seems some intronic miRNAs (intragenic) use unique promtors, which are different from the host promoters. What kind of experiments I can do to test if they share the same promoter? I detected endogenous miRNA expression in cancer cell lines by RT-PCR. Some cell lines the expression level is very high. Thanks again for kind help.

Intergenic is different to intronic. But anyway, have you checked expression of any endogenous miRNA in your cell line? Some cancers and perhaps cancer cell lines have mutations in the miRNA biogenesis machinery.

As to Jon's questions, perhaps the Drosha processing isn't very efficient and so not all of the transcript is processed to pre-miRNA, leaving some of the host mRNA intact? I suppose though that if the lariat is long enough and has the correct secondary structure and/or Drosha recognition sequence then it could be precessed. As far as I know (and please correct me) the recognition sequence for Drosha hasn't been determined? If we knew that, it might be a lot easier to understand!

[pcrman]

First you can do some bioinformatics analysis to see if you can find if the sequence upstream of the miRNA has promoter features such as CpG island, TATA box, etc.

If the miRNA uses the promoter of its host gene, they may be coexpressed. So check their expression in different cell lines may help. Can the transcription of the host gene be manipulated by some treatment? if so, treat your cells to see if the expression of the miRNA also changes.

[j123]

That's a very good idea. I am trying to treat cells to check the miRNA expression. Thanks a lot.

First you can do some bioinformatics analysis to see if you can find if the sequence upstream of the miRNA has promoter features such as CpG island, TATA box, etc.

If the miRNA uses the promoter of its host gene, they may be coexpressed. So check their expression in different cell lines may help. Can the transcription of the host gene be manipulated by some treatment? if so, treat your cells to see if the expression of the miRNA also changes.