Different lenght of 3-UTR (spliced?) - (Dec/14/2010 )
I have got the following problem: I harvested RNA from infected cells (DNA virus), transcribed it, using a 3-UTR adapter from Ambion into cDNA and did a PCR with gene specific primers, with the goal to check for the size of my 3-UTR of my viral mRNA. I got one relative nice band and I used this product for cloning into the Topo-vector. I picked different clones and analysed them via digest. What I got were clones having different insert length (shorter than expected compared to my PCR product). When I sequenced them, I discovered, that they contained parts of my gene (where my PCR primer started) and then different length of the 3-prime UTR region. Suprisingly some clones had 3-UTR which looked like they were spliced. Meaning that 100 of bp were missing before the sequence continued. However, when I checked the sequence, I could not find any usual splicing sites (eg GT-AG rule or AT-AC rule). What does that mean? Can it still be that the 3-UTR is spliced? But if so, why do I not have the usual intron-exon sequence? I rather get, just to name one example: 5'-Exon(ttc)-Intron(AGCNTTCC)-CCCTTT(Exon)-3' (Whereas the hyphen represents the part where the sequence is spliced).Non of the products I got follows the rule. However all clones I got have the expected sequence and also contain the polyA-tail. It is driving my nuts. Unfortunately, I have just started diving into the the potential spliced versions have a "normal splicing site", maybe I have overlooked something. I hope someone can help me, so therefore already thanks a lot.
I don't know if this is the case in your samples, but I know that some 3' UTRs go through an "alternative cleavage and polyadenylation" especially in a cancer cell line. I don't know if that'd happen in a bacteria or not though... Still, you may want to check this paper out: http://www.ncbi.nlm.nih.gov/pubmed/19703394
If your 3' UTR has several polyadenylation signals (AAUAAA, most commonly but also AUUAAA, AAGAAA, UAUAAA, or AGUAAA) it may be causing the alternative cleavage.