Poly(A) Binding Protein C1 Is Essential for Efficient L1 Retrotransposition and Affects L1 RNP Formation

Abstract

Poly(A) binding proteins (PABPs) specifically bind the polyadenosine tail of mRNA and have been shown to be important for RNA polyadenylation, translation initiation, and mRNA stability. Using a modified L1 retrotransposition vector, we examined the effects of two PABPs (encoded by PABPN1 and PABPC1) on the retrotransposition activity of the L1 non-long-terminal-repeat (non-LTR) retrotransposon in both HeLa and HEK293T cells. We demonstrated that knockdown of these two genes by RNA interference (RNAi) effectively reduced L1 retrotransposition by 70 to 80% without significantly changing L1 transcription or translation or the status of the poly(A) tail. We identified that both poly(A) binding proteins were associated with the L1 ribonucleoprotein complex, presumably through L1 mRNA. Depletion of PABPC1 caused a defect in L1 RNP formation. Knockdown of the PABPC1 inhibitor PAIP2 increased L1 retrotransposition up to 2-fold. Low levels of exogenous overexpression of PABPN1 and PABPC1 increased L1 retrotransposition, whereas unregulated overexpression of these two proteins caused pleiotropic effects, such as hypersensitivity to puromycin and decreased L1 activity. Our data suggest that PABPC1 is essential for the formation of L1 RNA-protein complexes and may play a role in L1 RNP translocation in the host cell. L ong interspersed element 1s (L1s or LINE-1s) are the most abundant autonomous non-long-terminal-repeat (non-LTR) human retrotransposons, accounting for ϳ17% of human DNA (45). Although most L1 copies are functionally inactive, there are ϳ80 to 100 retrotransposition-competent L1s in the human genome (9). L1s have had a great impact on shaping the human genome by their own retrotransposition and by mobilization of nonautonomous elements (Alu, SVA, and processed pseudogenes) in trans (17,18,21,32,60,64). Full-length L1 elements are ϳ6 kb long and contain a 5= untranslated region (5=UTR) and two nonoverlapping open reading frames (ORF1 and ORF2), followed by a short 3=UTR ending in a polyadenosine tail encoded, at least in part, in the DNA (5,20,23,24,31,66). The product of ORF1 is a 40-kDa protein (ORF1p) with nucleic acid binding and chaperone activities (35,36,39,40,51). ORF2 encodes an ϳ150-kDa multifunctional protein (ORF2p) with endonuclease (EN) (26) and reverse transcriptase (RT) activities (53, 62) and a cysteinerich domain of unknown function (22).L1 proliferates through a complex life cycle beginning with RNA polymerase II (Pol II) transcription of its mRNA, which is then exported to the cytoplasm for translation. Both ORFs are translated in the cytoplasm and exhibit a strong cis preference, presumably via direct association with the mRNA molecule encoding them (21, 44, 72). They form a ribonucleoprotein (RNP) particle likely to be a direct retrotransposition intermediate that is then presumed to enter the nucleus in some form (34,43,50). Several lines of evidence suggest that L1 transposes via the mechanism known as target-primed reverse transcription (TPRT) (13,26), which is utilized by other retro...

The Influence of LINE-1 and SINE Retrotransposons on Mammalian Genomes

“…The knockdown of hnRNPL led to an increase in the levels of mouse LINE-1 RNA and ORF1p and an increase in the retrotransposition of engineered LINE-1 in cultured cells (346). In addition, the poly-A binding protein C1 (PABPC1) interacts with the LINE-1 RNA and is required for LINE-1 RNP formation and efficient retrotransposition (347). Finally, cell culture assays have shown that human LINE-1 and mouse IAP mobility are restricted by the RNase activity of human RNase L, a member of the 2’,5’-oligoadenylate (2–5A) synthetase (OAS)-RNase L system, initially described for restricting viral infections during the interferon antiviral response (348).…”

Regulation of LINE-1 in mammals

“…Moreover, knockdown experiments of the corresponding mRNA result in significant reduction of L1 retrotransposition rate. By opposition, in PAIP2 knockdown cells, an inhibitor of PABPC1, a 2-fold L1 retrotransposition increase has been observed (136). Taken together, these results suggest that PABPC1 is essential for the formation of the RNP, and any depletion of PABPC1 can lead to defect in the RNP formation and in the achievement of a retrotransposition cycle (136).…”

“…By opposition, in PAIP2 knockdown cells, an inhibitor of PABPC1, a 2-fold L1 retrotransposition increase has been observed (136). Taken together, these results suggest that PABPC1 is essential for the formation of the RNP, and any depletion of PABPC1 can lead to defect in the RNP formation and in the achievement of a retrotransposition cycle (136). As it is known that PABPC1 can shuttle between the cytoplasm and the nucleus, the authors assumed that PABPC1 might provide a direct trafficking function for the RNP nuclear translocation, an essential step to complete a retrotransposition cycle (136).…”

“…Furthermore, in human cells, Boeke's team showed that two poly(A) binding proteins (PABPs), PABPN1 and PABPC1, can affect the L1 retrotransposition activity (136). They proved that both of them are associated with L1 RNP complexes, probably through the ability of both proteins to bind the L1 mRNA.…”

Expression and detection of LINE-1 ORF-encoded proteins

“…By applying the same technique to other ORF1p/ORF2p mutants, we should be able to further dissect the L1-host interactome and catalog the relationships with respect to specific step(s) of the L1 lifecycle. A complementary approach includes coupling large scale RNAi with L1 induction in suspension cell culture, since we have been able to easily generate shRNA-L1 co-expression vectors 38 . This will allow us to test which L1 RNP constituents affect the remaining complement of proteins’ abilities to co-purify with the L1 RNP, as well as to assay effects on retrotransposition activity in vivo and in vitro.…”