Internal Polyadenylation of the Parvovirus B19 Precursor mRNA Is Regulated by Alternative Splicing

Wuxiang Guan,Qinfeng Huang,Fang Cheng,Jianming Qiu

doi:10.1074/jbc.m111.227439

Abstract

Alternative processing of parvovirus B19 (B19V) pre-mRNA is critical to generating appropriate levels of B19V mRNA transcripts encoding capsid proteins and small nonstructural proteins. Polyadenylation of the B19V pre-mRNA at the proximal polyadenylation site ((pA)p), which prevents generation of full-length capsid proteins encoding mRNA transcripts, has been suggested as a step that blocks B19V permissiveness. We report here that efficient splicing of the B19V pre-mRNA within the first intron (upstream of the (pA)p site) stimulated the polyadenylation; in contrast, splicing of the B19V pre-mRNA within the second intron (in which the (pA)p site resides) interfered with the polyadenylation, leading to the generation of a sufficient number of B19V mRNA transcripts polyadenylated at the distal polyadenylation site ((pA)d). We also found that splicing within the second intron and polyadenylation at the (pA)p site compete during processing of the B19V pre-mRNA. Furthermore, we discovered that the U1 RNA that binds to the 5' splice donor site of the second intron is fully responsible for inhibiting polyadenylation at the (pA)p site, whereas actual splicing, and perhaps assembly of the functional spliceosome, is not required. Finally, we demonstrated that inhibition of B19V pre-mRNA splicing within the second intron by targeting an intronic splicing enhancer using a Morpholino antisense oligonucleotide prevented B19V mRNA transcripts polyadenylated at the (pA)d site during B19V infection of human erythroid progenitors. Thus, our study reveals the mechanism by which alternative splicing coordinates alternative polyadenylation to generate full-length B19V mRNA transcripts at levels sufficient to support productive B19V infection.

Highlights

We found that Ͼ95% of the mRNA transcripts generated from the pCI(pA)p400 and pCI(pA)p240 read through thep site (Fig. 2B, lanes 2 and 3)
Similar to what we observed from transfection of these mutants in the context of B19V replication-competent backbone in COS-7 cells, we found that an increase in splicing of the second intron at the A2-1 site improved B19V pre-mRNA read through thep site significantly (ratio ofp/(pA)d was decreased from 1 to Ͻ0.1; Fig. 4D, lane 12) and that knock-out of splicing of the central intron prevented B19V pre-mRNA read through thep site. (The ratio ofp/(pA)d was increased from 1 to 10; Fig. 4D, lane 13.)
We analyzed how alternative polyadenylation of the B19V pre-mRNA is regulated by its alternative splicing in the context of a replication-competent B19V genome in B19V nonpermissive COS-7 and B19V-permisisve UT7/Epo-S1 cells [20, 25, 26], as well as during B19V infection of ex vivo-expanded primary erythroid progenitor cells

Summary

Introduction

Cell aplasia (infection of immunocompromised patients), and hydrops fetalis (infection of pregnant women). Constructs to Analyze Significance of U1 RNA Binding in Polyadenylation at (pA)p—Plasmid C1B19ISE2A2-1KO was constructed from the parent plasmid C1NS1(Ϫ) by mutating the G-rich intronic splicing enhancer (ISE) (nt 2371–2400) to 5Ј-CCGCGGCACGAGATCATCACGATCGAACAG-3Ј as described previously [18] and the A2-1 site from AG to AA.

Results

Conclusion