Abstract
The heterogeneous nuclear ribonucleoprotein D family of proteins also known as AUF1 consists of four isoforms implicated in both nuclear and cytoplasmic functions. The AUF1 proteins are largely nuclear but also are found in the cytoplasm and are thought to undergo nucleocytoplasmic shuttling. The nucleocytoplasmic distribution and potential shuttling activity of the individual AUF1 isoforms have not been previously studied in detail. Therefore, we characterized the nucleocytoplasmic transport of each of the heterogeneous nuclear ribonucleoprotein D/AUF1 isoforms. All four AUF1 proteins were found to undergo rapid nucleocytoplasmic shuttling in a manner that is transcription-independent, carrier-mediated, and energy-requiring. Nucleocytoplasmic shuttling of the AUF1 proteins is shown to utilize a novel arrangement of nuclear import and export signals. Mutagenesis of the AUF1 proteins and fusion of polypeptides to a green fluorescent protein reporter demonstrated that a nuclear import signal is located in the C-terminal domain of the protein and is found only in the two smaller isoforms. Further mapping demonstrated that nuclear export is facilitated by sequences in AUF1 exon 7 found in the C-terminal domain of the two larger AUF1 isoforms. A subset of AUF1 proteins are shown to directly interact in vitro using purified recombinant proteins and in vivo in the absence of RNA. These results suggest that nuclear import of AUF1 is facilitated by sequences found only in the two smaller isoforms and that nuclear export is facilitated by sequences (exon 7 and the C-terminal domain) found only in the two larger isoforms. This novel arrangement of signals might represent a mechanism to assure co-shuttling of a subset of AUF1 proteins that interact in a heterocomplex.
Highlights
Tin 1/CRM11 interacts directly with a leucine-rich nuclear export sequence in the cargo protein or with adapter molecules [1]
A subset of AUF1 proteins are shown to directly interact in vitro using purified recombinant proteins and in vivo in the absence of RNA. These results suggest that nuclear import of AUF1 is facilitated by sequences found only in the two smaller isoforms and that nuclear export is facilitated by sequences found only in the two larger isoforms
We found that the uninterrupted C-terminal domain (CTD) found in the p37 and p40 AUF1 isoforms possesses a strong nuclear import activity, whereas the CTD interrupted by exon 7 promotes cytoplasmic localization, which is inserted in the CTD of the p42 and p45 isoforms
Summary
Tin 1/CRM11 interacts directly with a leucine-rich nuclear export sequence in the cargo protein or with adapter molecules [1]. Facilitated nuclear import involves nuclear localization sequences (NLSs) within cargo proteins containing any of several canonical basic amino acid motifs [2]. Some cargo proteins utilize non-canonical NLSs for transport such as the M9 domain, which consists of a glycine-rich amino acid sequence that promotes both protein import and export, and functions as a shuttling sequence [3,4,5]. HnRNP A1 represents one type of shuttling protein that is a nuclear protein at steady state but shuttles rapidly and accumulates in the cytoplasm with transcription inhibition [3, 6], which is associated with the presence of an M9 domain [3]. HnRNP K is another type of shuttling RNA-binding protein It contains a canonical NLS in addition to a unique shuttling domain known as KNS. We show that a subset of AUF1 proteins can directly interact in vitro and in vivo in the absence of RNA, which suggests that nuclear import is probably facilitated by the p37 and p40 isoforms, whereas nuclear export is facilitated by the p42 and p45 isoforms as a part of a larger AUF1 complex of proteins
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