I am quite new with miRNAs and I have a few basic questions:
I am concerned about identifying the elements that drive the transcription of a miRNA precursor from a lentiviral plasmid. First of all, I want to know if what I have cloned in that plasmid is the pri-mir or the pre-mir, and also I would like to know if the pri-mir or pre-mir would be transcribed together with the reporter protein of the plasmid in only one primary transcrip, or by the contrary, the miRNA coding sequence has a motif to stop the transcription. In this later case two promotors will be needed for miRNA and GFP transcription, right?. But... I have seen some papers where miRNA and GFP are expresed by bicistronic vectors (IRES). How the transcription and translation would be in this type of plasmid? So, briefly, in first place I would like to know where to get the genomic secuence of the miR (so steem loop + flanking sequences). The NCBI Reference Sequence gives me only the stem loop sequence (the pre-mir).
Concerning the IRES site: IRES stands for "internal ribosome entry site". This means that for one promoter you get two transcripts, one for the miRNA, the other for the GFP. The IRES sequence itself is not transcribed.
To get the genomic sequence of the full length transcript out of Ensembl, let it search for e.g. mir205 (no dash added!) in human. This gives one gene, MIR205HG (HG = host gene). Click on this. Then you get the "Gene Summary" page. Click on "sequence" of the left menu panel. The sequence will be shown with exons and introns (if any) marked, and can be copied to your editor of choice.
If any specific transcript is needed: similar from the transcripts page.
I have done this through ensembl. it is a little tedious, but by looking at a miRNA's exon-view, you can see as many kb as you want of flanking sequence. the full length primary sequences are usually not annotated, however and you'd have to do something like RACE or sequencing based approach to find the true ends. As far as expression from a vector, probably your clone contains something more similar to the pri-miRNA. it doesn't need to be the full sequence, just enough to be recognized and entered into the biogenesis pathway. This way you do not have to include the Poly-A which terminates transcription. Your construct will include this anyway. Whether that is then done on a single transcript or multiple depends on your construct. ours typically use IRES-eGFP, giving a single transcript which is cleaved to produce the mature miRNA, as well as translated (via IRES) to produce eGFP. we are still able to detect eGFP in transduced cells, even though the transcripts are presumably being cleaved and destroyed.
Hi Maria
You should be able to find the genomic sequence information of the miRNA in mirbase database http://www.mirbase.org/
Cheers
Exactly, miRBase sequence of all human miRNA and many others. It has both stem loop sequence as well as mature sequence. I think you would be interested in pre-miR sequence. Now if you expect pri miRNA, then that information is not that easily available.
Hi Maria,
The GFF files or GFF3 files (general feature format, gene-finding format, generic feature format) describe information about genes and other features of DNA including the position of miRNA and pre-miRNAs in the genome sequence.
But one of the best DataBases for miRNAs is miRBase.
All the Best,
Matheus Gomes
Hi, it might not be the best way but I think you can get any flanking sequence by using a combination of miR BASE and Genome Browser. For example, you can get pre-miR sequence from miR BASE, then you can go to BLAT search of Genome Browser. You can copy and past your miRNA sequence and just do BLAT. You will see your miRNA loci on screen (you have to choose sno/miRNA option as full, there are many options below). If you have seen the correct your miRNA on screen (it could be red colour) you can zoom out/move your view as you want. When you get good view for you you can get the sequence of your view by clicking "View"->"DNA"->"get DNA".
This is quit manual but much more flexible and works well on me. One option.
You can also obtain them from Table browser of Genome browser or ENCODE project.
Ensembl would be my method of choice to get the complete transcript, if this is known. If you want to express a protein and an RNA from the same expression vector, an IRES-containing vector is indeed the solution. Put the reporter (GFP) behind the IRES sequence, and the gene of interest (i.e., your miRNA) in front of it, the position behind the IRES site tends to give the weaker expression.
It may not be necessary to clone the entire "host gene" of the miRNA (often several kb) into your expression vector, but I remember with the miR34a, a 3.6kb fragment gave a better expression than an1.8kb (the whole thing is about 5kb).
Hope this helps.
Hi there! Thank you for the help. Special thanks to Belinda that solved my doubt about miR expresion in a bicistronic vector. This means that miRNA + IRES + GFP is transcribed all together in the same primary transcritp right? How the miRNA is processed from there? Is the steem loop directly recognized and scised by Dicer in that primary transcript leaving the IRES and GFP behind?.
Indeed I went to www.mirbase.org before posting this question but I can not find where to get the genomic sequence from this data base. I easily find the steem loop sequence (pre-mir) and the mature sequence of the miRNA and miRNA* but not the genomic sequence. I am concerned about the regulatory signals in the genomic sequence (promotor and stop transcription signal). At the mirbase some miRs have a link to ensembl at the "genomic context" menu, but some not. Coud this be because the genome context of this particular miRNA is not known?
One final quenstion: once at Ensembl how do I get the FASTA sequence of the miR genomic sequence? Thank! ^^
Concerning the IRES site: IRES stands for "internal ribosome entry site". This means that for one promoter you get two transcripts, one for the miRNA, the other for the GFP. The IRES sequence itself is not transcribed.
To get the genomic sequence of the full length transcript out of Ensembl, let it search for e.g. mir205 (no dash added!) in human. This gives one gene, MIR205HG (HG = host gene). Click on this. Then you get the "Gene Summary" page. Click on "sequence" of the left menu panel. The sequence will be shown with exons and introns (if any) marked, and can be copied to your editor of choice.
If any specific transcript is needed: similar from the transcripts page.
From mirBase, there are sevral ways to extract the genomic context, see information at http://www.mirbase.org/help/genome.shtml
Can you give us an example without genomic coordinates because I don't find a case like this? All miRNAs should be mappable to the genomic sequence and should have a genomic context, but it is possible that annotations such as TSS and IRES are missing.
To get the genomic sequence from Ensembl, I would zoom with the genome browser to the region of intrerest (you can click on a miRNA location link) and then use "Export Data" from the left menu. You should be able to export the sequence in fasta format.
get the genomic sequence from data baseso easily find the steem loop sequence (pre-mir) and the mature sequence of the miRNA and miRNA* but not the genomic sequence.and there a kid
Check out http://www.nature.com/natureevents/science/events/47533-microRNA_Analysis_Using_Next_Generation_Sequencing
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