(637) mRNA Binding Proteins PTBP1 and HNRNPL Modulate CFTR mRNA Abundance

Background: 10% of cystic fibrosis (CF) patients carry an in-frame premature termination codon (PTC) in the CFTR mRNA. A PTC leads to negligible CFTR protein expression/function because: 1) a PTC often triggers nonsense-mediated mRNA decay (NMD), resulting in less mRNA for translation; and 2) for mRNAs that escape NMD, the PTC terminates translation before a full-length, functional protein is made. Thus, CF patients who carry a PTC do not benefit from current CFTR modulator drugs that target CFTR protein. Several treatments are under development to treat CF patients who carry PTCs, including therapies that suppress translation termination at PTCs using low molecular weight compounds or suppressor tRNAs, or mRNA mutation repair therapies. However, the effectiveness of these therapies is limited by CFTR mRNA abundance, which is reduced between 2- to 20-fold by NMD. NMD is categorized into two types: 1) classical exon-junction complex (EJC) mediated NMD and 2) non-classical 3’ UTR mediated NMD. For classical NMD, a subset of NMD factors associate with EJCs that become bound to mRNAs near exon-exon junctions during splicing. If a PTC lies at least 50-55 nucleotides upstream of an EJC, PTC-bound NMD factors can interact with EJC-bound NMD factors to initiate mRNA decay. For non-classical NMD, the NMD factor UPF1 accumulates on long 3’ UTRs to trigger mRNA decay. Because human CFTR mRNA carries a long, 1.6 kilobase 3’ UTR, it may undergo 3’ UTR mediated NMD. CFTR mRNAs containing PTCs may be subject to both EJC and 3’ UTR mediated NMD; wild-type (WT) CFTR mRNA may undergo 3’ UTR mediated NMD. Recently, mRNA binding proteins HNRNPL and PTBP1 were shown to compete with UPF1 for binding to the 3’ UTR of certain mRNAs and protect them from NMD. The CFTR 3’ UTR contains multiple CA and CU rich regions that may serve as HNRNPL or PTBP1 binding sites, respectively. We hypothesize that HNRNPL and/or PTBP1 bind to the 3’ UTR of CFTR mRNAs and protect them from NMD.

Methods: To examine whether HNRNPL and PTBP1 alter CFTR mRNA abundance, we transfected siRNAs into wild-type and gene-edited CFTR G542X and W1282X 16HBE14o- (16HBE) cells to knock down HNRNPL and PTBP1, which was verified using qPCR and immunoblotting. Using qPCR, CFTR mRNA levels were quantitated in each 16HBE cell line after transfection of siRNAs to HNRNPL or PTBP1 relative to controls.

Results: 16HBEs transfected with siRNAs to HNRNPL or PTBP1 showed a 50-60 percent reduction in both mRNA and protein abundance compared to cells transfected with a control siRNA, indicating efficient knockdown of both mRNA binding proteins. CFTR mRNA abundance in 16HBEs transfected with an HNRNPL siRNA compared to the control siRNA was increased in both G542X and W1282X 16HBEs, but not in wild-type 16HBEs. Transfection of 16HBEs with a PTBP1 siRNA showed an increase in CFTR mRNA abundance in wild-type, G542X, and W1282X 16HBEs relative to cells transfected with the control siRNA.

Conclusions: HNRNPL depletion increased the abundance of CFTR mRNAs carrying PTCs. This suggests HNRNPL primarily modulates EJC mediated NMD. However, PTBP1 depletion increased the abundance of both wild-type and PTC-containing CFTRmRNAs, suggesting PTBP1 modulates 3’ UTR mediated NMD. Other mechanisms are also possible. Overall, these results suggest that both HNRNPL and PTBP1 modulate CFTR mRNA abundance and represent targets to enhance the effectiveness of therapies directed at PTCs.

Acknowledgements: This work was supported by the Cystic Fibrosis Foundation (KEEL20G0).

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