The threonine residues in the N-terminus regulatory domain are conserved together with the surrounding sequence.
This glutamic acid residue and its surrounding sequence have been conserved during eukaryotic evolution, suggesting a functional importance.
Interestingly the R102Q mutation reduced the α-helical propensity of surrounding sequence by approximately four-fold (Table S1).
The motif copies are significantly more conserved than the surrounding sequence, which suggests that they play an important functional role.
We also checked for a few "recently gained" intron positions (25a, 27, 29, 53b pooled) whether the surrounding sequence showed a consensus motif.
The Ac-DAF-16 DBD bound to amplicons containing this element, but not individual oligos, suggesting that the new element requires surrounding sequence for DBD binding.
One of these, SIM2, has several acidic residues in the surrounding sequence, as well as a consensus CK2 phosphorylation site, although the importance of this remains to be tested.
Finally those reads which contained additional uridines, but the surrounding sequence matched 100% to the mature miRNA (similar to an insertional polymorphism) were identified as miRNAs affected by internal uridylation.
For each of these candidates the surrounding sequence was assessed to determine if it was able to form a hairpin structure using the Vienna RNAfold package [22], indicative of a pre-miRNA.
If the surrounding sequence was able to form a hairpin structure using the Vienna RNAfold package [22], indicative of a pre-miRNA, then the unique sequence was deemed to be a novel candidate miRNA.
Oldest introns (positions 1, 5, 11) showed a good fit to the sequence AG/GT, whereas we found gained intron positions for which the surrounding sequence was completely different (Table S2).
