Identification of charged amino acids required for nuclear localization of human L1 ORF1 protein

Abstract

Background Long Interspersed Element 1 (LINE-1) is a retrotransposon that is present in 500,000 copies in the human genome. Along with Alu and SVA elements, these three retrotransposons account for more than a third of the human genome sequence. These mobile elements are able to copy themselves within the genome via an RNA intermediate, a process that can promote genome instability. LINE-1 encodes two proteins, ORF1p and ORF2p. Association of ORF1p, ORF2p and a full-length L1 mRNA in a ribonucleoprotein (RNP) particle, L1 RNP, is required for L1 retrotransposition. Previous studies have suggested that fusion of a tag to L1 proteins can interfere with L1 retrotransposition. Results Using antibodies detecting untagged human ORF1p, western blot analysis and manipulation of ORF1 sequence and length, we have identified a set of charged amino acids in the C-terminal region of ORF1p that are important in determining its subcellular localization. Mutation of 7 non-identical lysine residues is sufficient to make the resulting ORF1p to be predominantly cytoplasmic, demonstrating intrinsic redundancy of this requirement. These residues are also necessary for ORF1p to retain its association with KPNA2 nuclear pore protein. We demonstrate that this interaction is significantly reduced by RNase treatment. Using co-IP, we have also determined that human ORF1p associates with all members of the KPNA subfamily. Conclusions The prediction of NLS sequences suggested that specific sequences within ORF1p could be responsible for its subcellular localization by interacting with nuclear binding proteins. We have found that multiple charged amino acids in the C-terminus of ORF1p are involved in ORF1 subcellular localization and interaction with KPNA2 nuclear pore protein. Our data demonstrate that different amino acids can be mutated to have the same phenotypic effect on ORF1p subcellular localization, demonstrating that the net number of charged residues or protein structure, rather than their specific location, is important for the ORF1p nuclear localization. We also identified that human ORF1p interacts with all members of the KPNA family of proteins and that multiple KPNA family genes are expressed in human cell lines. Electronic supplementary material The online version of this article (10.1186/s13100-019-0159-2) contains supplementary material, which is available to authorized users.

Factors Regulating the Activity of LINE1 Retrotransposons

“…It contains a bi-directional promoter in the 5′UTR; two open reading frames, namely ORF1 and ORF2, and a 3′UTR with a polyadenylation signal (polyA) (Figure 1) [12][13][14]. ORF1 encodes a ~40 kD protein with the chaperone activity necessary to stabilize a new L1 copy [15][16][17][18]. ORF2 encodes ~150 kD proteins with the endonuclease and retrotransposase activity required for the retrotransposition process [15,19,20].…”

“…Transportation of L1 RNP from the cytoplasm to the nucleus is accomplished using the membrane-associated endosomal sorting complex required for transportation (ESCRT) [30]. It has also recently been shown that the ORF1 protein interacts with the KPNA2 and KPNB1 karyopherins, as well as possibly other KPNA family proteins involved in nuclear protein importation through nuclear pores (nuclear pore complex) [18]. The cancer cell model showed that L1 RNP penetrates the nucleus during mitosis and the integration of the new copy of the L1 element into the genome occurs in the S phase of a cell cycle [31].…”

“…The structure of a full-length copy of L1 retrotransposon. ORF1 consists of an N-terminal domain (N), a coiled-coil domain (CCD), an RNA recognition motive (RRM), and a C-terminal domain (CTD) [18]. ORF2 consists of endonuclease (EN), retrotransposase (RT), a cryptic domain (Cry), a Z-domain (Z), and a C-terminal domain with a cysteine-rich region (Cys-rich) [24].…”

Bird Eye View of Protein Subcellular Localization Prediction

“…The limitation of homology-based methods is more pronounced in cases where no homology is found between the query sequence and the annotated proteins sequence. Additionally, it is known that a single amino acid substitution in localization signals can change the localization of a protein [ 25 , 26 , 27 ]. Thus, sequence homology is a noncausal feature for the localization prediction and should be used with caution when applied to nonnative sequences or in case when homology is less [ 28 ].…”

Enterovirus Infection Restricts Long Interspersed Element 1 Retrotransposition

“…L1 proteins interact with L1 RNA to facilitate the assembly of L1 ribonucleoproteins (RNPs). The entry process by which L1 RNPs move from the cytoplasm into the nucleus is dependent on the nuclear localization signal on L1 ORF1p ( Freeman et al, 2019 ). Upon entering the nucleus, L1 ORF2p uses its nuclease activity to form a single chain gap on genome DNA, and then uses its RT activity, with the single chain gap DNA as the primer and L1 mRNA as the template, to start the reverse transcription ( Kazazian and Moran, 1998 ; Goodier, 2016 ).…”