miRNA 3' end nucleotidylation - (Sep/29/2008 )
Several large sequencing studies have found that mature miRNAs contain untemplated nucleotides at their 3' termini. I have tried to clone and sequence specific miRNAs, rather then do library cloning, and found in some cases some miRNAs have nongenomic uridines and adenines. The problem with these results is that the findings are not consistent and I'm getting different results each time I run the experiment. To clone I use the 3' adaptor approach outlined by the Bartel protocol, perform RT, and then PCR using an upstream primer that matches the mature miRNA of interest. The PCR products are precipitated and then TA cloned for sequencing. I would appreciate any insight or ideas on how I could address this issue. Thanks.
What results do you get? ... sometimes there are extra nucleotides and sometimes there aren't? Different length and composition of added tails? Have you tried your cloning protocol with different miRNAs and starting from different tissues?
hi andrea,
the results are all over the place. i'm using a549 cells and i find that the composition of the specific mature miRNA tails are either a couple extra (1-3 nt) untemplated adenines or uridines (or mix of the two). what is constant is that there are almost never Cs or Gs present. i have not tried other cell lines or tissues yet.
Hi Mateo,
as it has been reported several times and resulting from different cloning approaches, nucleotide addition at the 3' end of miRNAs is unlikely to be a cloning artefact. Maybe, to be sure, you could try to detect the different forms by northern. Also, there are indications that the As and Us are added enzymatically, though, as far as I know, the enzymes are not identified. The 3' modifications may affect miRNA stability/degradation. In Arabidopsis an exoribonuclease involved in miRNA degradation has been identified (Ramachandran and Chen, science, september 2008), and it's activity has been shown to be reduced by untemplated Us at miRNA 3' end. If in the cell line you're using the proportion of miRNAs with 3' additions is greater than previously reported, I think it would be intersting to check their stability in comparison with unmodified miRNAs. I work on plants, and know very little about the animal systems, so that you may find quite naive these comments. But I wish someone working in your field will conbtribute more properly on this topic.
Andrea
i think this is the result of sequence, as we know the special structure is difficult to sequence. 
as it has been reported several times and resulting from different cloning approaches, nucleotide addition at the 3' end of miRNAs is unlikely to be a cloning artefact. Maybe, to be sure, you could try to detect the different forms by northern. Also, there are indications that the As and Us are added enzymatically, though, as far as I know, the enzymes are not identified. The 3' modifications may affect miRNA stability/degradation. In Arabidopsis an exoribonuclease involved in miRNA degradation has been identified (Ramachandran and Chen, science, september 2008), and it's activity has been shown to be reduced by untemplated Us at miRNA 3' end. If in the cell line you're using the proportion of miRNAs with 3' additions is greater than previously reported, I think it would be intersting to check their stability in comparison with unmodified miRNAs. I work on plants, and know very little about the animal systems, so that you may find quite naive these comments. But I wish someone working in your field will conbtribute more properly on this topic.
Andrea