Key result
U1-70K mediates inhibition of AAV5 internal polyadenylation, and interaction with the cap binding complex strengthens U1 snRNP binding to the downstream intron donor in a distance-dependent manner.
The study demonstrates that U1-70K and the cap binding complex strengthen U1 snRNP binding to the downstream intron donor, inhibiting polyadenylation at the AAV5 (pA)p site in a distance-dependent manner.
May guide AAV vector optimization for gene therapy; leaves open clinical translation to cardiovascular applications.
Adeno-associated virus type 5 (AAV5) is unique among human AAV serotypes in that it uses a polyadenylation site [(pA)p] within the single small intron in the center of the genome. We previously reported that inhibition of polyadenylation at (pA)p, necessary for read-through of P41-generated capsid gene pre-mRNAs which are subsequently spliced, requires binding of U1 snRNP to the upstream donor. Inhibition was reduced as the distance between the cap site and the donor was increased (increasing the size of the 5' exon). Here, we have demonstrated that U1-70K is a key component of U1 snRNP that mediates inhibition of polyadenylation at (pA)p. Furthermore, introduction of a U-rich stretch, predicted to target TIA-1 and thus increase the affinity of U1 snRNP binding to the intervening donor site, significantly augmented inhibition of (pA)p, while depletion of TIA-1 by siRNA increased (pA)p read-through. Finally, artificially tethering the cap binding complex (CBC) components CBP80 and CBP20 upstream of the intron donor increased inhibition of polyadenylation at (pA)p. Our results suggest that interaction with the CBC strengthens U1 snRNP binding to the downstream intron donor in a manner inversely proportional to the size of the 5' exon, thus governing the competition between intron splicing and polyadenylation at (pA)p. This competition must be optimized to program both the levels of polyadenylation of P7- and P19-generated RNA at (pA)p required to produce proper levels of the essential Rep proteins and the splicing of P41-generated RNAs to produce the proper ratio of capsid proteins during AAV5 infection.
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Qiu et al. (2007) studied AAV5 RNA processing (in vitro). U1-70K overexpression, TIA-1 modulation, and CBC retargeting vs. Control plasmids or random siRNA was evaluated on Polyadenylation of AAV5 pre-mRNAs at the internal site (pA)p. U1-70K mediates inhibition of AAV5 internal polyadenylation, and interaction with the cap binding complex strengthens U1 snRNP binding to the downstream intron donor in a distance-dependent manner.
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