Hey all,
To overexpress a miRNA; instead of pre-miRNA sequences, pri-miRNA sequences are also being used driven by RNAP II promoters.
in articles these fragments nearly miRNA+100-300 bp flanking sequences. Can a whole fragment with 1 kb containing pre-miRNA be used? Or the long RNA transcript may esgape from drosha processing?
Is there any limitations of pri-miRNA lenghts except RNAP transcription capacity?
Hi Enes,
In general, yes, 1 kb fragment can be used to overexpress a miRNA of interest. But there are few exceptions, that flanking segments may contain cis-element(s) inhibitory for Drosha processing. For example, please refer to the following paper: A Biogenesis Step Upstream of Microprocessor Controls miR-17∼92 Expression. Cell (2015)
Just an update,
I got my experiment results. The microRNA is upregulated with this cloning.
hi Research Brothers, i need help, what is the exact starting point of pri-miRNA? and also approximate length of pri-miRNA.
hi,
you can check it on NCBI.
NCBI >select "GENE"> search for miRNA such as "Hsa-mir-96"
you will get the exact sequence of pre-miRNA including start nucleotide.
Any nucleotide added more, creates pri-miRNA such as pri-miR96 contains a contig with miR182 and miR183 a huge contig that contains 3 miRNA is actually a pri-miRNA sequence. so there is no approximate lenght.
The more, an intron that contains a miRNA is a form of pri-miRNA which will be further cleaved in to pre-miRNA.
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