Sam68 is absolutely required for Rev function and HIV-1 production

Sam68 functionally complements for, as well as synergizes with, HIV-1 Rev in Rev response element (RRE)-mediated gene expression and virus production. Furthermore, C-terminal deletion/point mutants of Sam68 (Sam68ΔC/Sam68-P21) exert a transdominant negative phenotype for Rev function and HIV-1 production. However, the relevance of Sam68 in Rev/RRE function is not well defined. To gain more insight into the mechanism of Sam68 in Rev function, we used an RNAi (RNA interference) strategy to create stable Sam68 knockdown HeLa (SSKH) cells. In SSKH cells, Rev failed to activate both RRE-mediated reporter gene [chloramphenicol acetyltransferase (CAT) and/or gag] expressions. Importantly, reduction of Sam68 expression led to a dramatic inhibition of HIV-1 production. Inhibition of the reporter gene expression and HIV production correlated with the failure to export RRE-containing CAT mRNA and unspliced viral mRNAs to the cytoplasm, confirming that SSKH cells are defective for Rev-mediated RNA export. Taken together, these results suggest that Sam68 is involved in Rev-mediated RNA export and is absolutely required for HIV production.     

Sam68 RNA binding protein is an in vivo substrate for protein arginine N-methyltransferase 1

RNA binding proteins often contain multiple arginine glycine repeats, a sequence that is frequently methylated by protein arginine methyltransferases. The role of this posttranslational modification in the life cycle of RNA binding proteins is not well understood. Herein, we report that Sam68, a heteronuclear ribonucleoprotein K homology domain containing RNA binding protein, associates with and is methylated in vivo by the protein arginine N-methyltransferase 1 (PRMT1). Sam68 contains asymmetrical dimethylarginines near its proline motif P3 as assessed by using a novel asymmetrical dimethylarginine-specific antibody and mass spectrometry. Deletion of the methylation sites and the use of methylase inhibitors resulted in Sam68 accumulation in the cytoplasm. Sam68 was also detected in the cytoplasm of PRMT1-deficient embryonic stem cells. Although the cellular function of Sam68 is unknown, it has been shown to export unspliced human immunodeficiency virus RNAs. Cells treated with methylase inhibitors prevented the ability of Sam68 to export unspliced human immunodeficiency virus RNAs. Other K homology domain RNA binding proteins, including SLM-1, SLM-2, QKI-5, GRP33, and heteronuclear ribonucleoprotein K were also methylated in vivo. These findings demonstrate that RNA binding proteins are in vivo substrates for PRMT1, and their methylation is essential for their proper localization and function.     

Inhibition of HIV replication by dominant negative mutants of Sam68, a functional homolog of HIV-1 Rev.

The HIV-1 Rev protein facilitates the nuclear export of mRNA containing the Rev response element (RRE) through binding to the export receptor CRM-1. Here we show that a cellular nuclear protein, Sam68 (Src-associated protein in mitosis), specifically interacts with RRE and can partially substitute for as well as synergize with Rev in RRE-mediated gene expression and virus replication. Differential sensitivity to leptomycin B, an inhibitor of CRM-1, indicates that the export pathways mediated by Rev and Sam68 are distinct. C-terminally deleted mutants of Sam68 inhibited the transactivation of RRE-mediated expression by both wild-type Sam68 and Rev. They were retained in the cytoplasm and impeded the nuclear localization of Rev in co-expressed cells. These mutants also inhibited wild-type HIV-1 replication to the same extent as the RevM10 mutant, and may be useful as anti-viral agents in the treatment of AIDS.     

Identification of Sam68 arginine glycine-rich sequences capable of conferring nonspecific RNA binding to the GSG domain

Sam68 is an RNA-binding protein that contains a heterogeneous nuclear ribonucleoprotein K homology domain embedded in a larger RNA binding domain called the GSG (GRP33, Sam68, GLD-1) domain. This family of proteins is often referred to as the STAR (signal transduction and activators of RNA metabolism) proteins. It is not known whether Sam68 is a general nonspecific RNA-binding protein or whether it recognizes specific response elements in mRNAs with high affinity. Sam68 has been shown to bind homopolymeric RNA and a synthetic RNA sequence called G8-5 that has a core UAAA motif. Here we performed a structure function analysis of Sam68 and identified two arginine glycine (RG)-rich regions that confer nonspecific RNA binding to the Sam68 GSG domain. In addition, by using chimeric proteins between Sam68 and QKI-7, we demonstrated that one of the Sam68 RG-rich sequences of 26 amino acids was sufficient to confer homopolymeric RNA binding to the GSG domain of QKI-7, another STAR protein. Furthermore, that minimal sequence can also give QKI-7 the ability (as Sam68) to functionally substitute for HIV-1 REV to facilitate the nuclear export of RNAs. Our studies suggest that neighboring RG-rich sequences may impose nonspecific RNA binding to GSG domains. Because the Sam68 RNA binding activity is negatively regulated by tyrosine phosphorylation, our data lead us to propose that Sam68 might be a specific RNA-binding protein when tyrosine phosphorylated.     

Arginine methylation of Sam68 and SLM proteins negatively regulates their poly(U) RNA binding activity

Sam68 (Src substrate associated during mitosis) and its homologues, SLM-1 and SLM-2 (Sam68-like mammalian proteins), are RNA binding proteins and contain the arg-gly (RG) repeats, in which arginine residues are methylated by the protein arginine methyltransferase 1 (PRMT1). However, it remains unclear whether the arginine methylation affects an RNA binding. Here, we report that methylation of Sam68 and SLM proteins markedly reduced their poly(U) binding ability in vitro. The RG repeats of Sam68 bound poly(U), but arginine methylation of the RG repeats abrogated its poly(U) binding ability in vitro. Overexpression of PRMT1 increased arginine methylation of Sam68 and SLM proteins in cells, which resulted in a decrease of their poly(U) binding ability. The results suggest that the RG repeats conserved in Sam68 and SLM proteins may function as an auxiliary RNA binding domain and arginine methylation may eliminate or reduce an RNA binding ability of the proteins.     

Expression of exogenous Sam68, the 68-kilodalton SRC-associated protein in mitosis, is able to alleviate impaired Rev function in astrocytes

Human immunodeficiency virus type 1 (HIV-1) gene expression in astrocytes is restricted, resulting in a brief and limited synthesis of HIV-1 viral structural proteins. Impaired Rev function has been documented in these cells. However, the molecular mechanisms underlying the impaired Rev function are not fully understood. Using the astroglial cell line U87.MG as a model, we report here that HIV-1 gene expression down-regulated expression of Sam68, the 68-kDa Src-associated protein in mitosis, which was constitutively expressed at a lower level in astrocytes. Elevating the endogenous level of Sam68 expression considerably restored HIV-1 Rev function in astrocytes, as determined by a Rev-dependent reporter gene assay. However, elevation of Sam68 expression achieved only a modest increase in HIV-1 production, further supporting the notion that there are multiple cellular restrictions of HIV-1 gene expression in astrocytes. Mutagenesis analysis identified the region between amino acids 321 and 410 of Sam68 as being directly involved in the binding of Sam68 to Rev, while a double mutation in Rev, L78D and E79L, like those in the dominant-negative Rev mutant M10, eliminated Rev binding to Sam68. Moreover, subcellular fractionation and digital fluorescence microscopic imaging revealed that Sam68 expression promoted Rev nuclear export. Taken together, our studies demonstrate that a lower level of constitutive Sam68 expression, followed by further down-regulation by HIV-1 infection, contributes to impaired Rev function in astrocytes, and they suggest that Sam68 may play an important role in Rev nuclear export.     

An anti-apoptotic protein, Hax-1, inhibits the HIV-1 rev function by altering its sub-cellular localization

The human immunodeficiency virus type 1 (HIV-1) Rev protein facilitates the nuclear export of viral mRNA containing the Rev response element (RRE). Although several host proteins co-operating with Rev in viral RNA export have been reported, little is known about the innate host defense factors that Rev overcomes to mediate the nuclear export of unspliced viral mRNAs. We report here that an anti-apoptotic protein, HS1-associated protein X-1 (Hax-1), a target of HIV-1 Vpr, interacts with Rev and inhibits its activity in RRE-mediated gene expression. Co-expression of Sam68 emancipates Rev activity from Hax-1-mediated inhibition. Hax-1 does not bind to RRE RNA by itself, but inhibits Rev from binding to RRE RNA in vitro. The impact of Hax-1 on Rev/RRE interactions in vitro correlates well with the reduced level of RRE-containing mRNA in vivo. Immunofluorescence studies further reveal that Hax-1 and Rev are cytoplasmic and nuclear proteins, respectively, when expressed independently. However, in Hax-1 co-expressing cells, Rev is translocated from the nucleus to the cytoplasm, where it is co-localized with Hax-1 in the cytoplasm. We propose that over-expression of Hax-1, possibly through binding to Rev, may interfere with the stability/export of RRE-containing mRNA and target the RNA for degradation.     

Self-association of the single-KH-domain family members Sam68, GRP33, GLD-1, and Qk1: role of the KH domain.

Sam68 is a member of a growing family of proteins that contain a single KH domain embedded in a larger conserved domain of approximately 170 amino acids. Loops 1 and 4 of this KH domain family are longer than the corresponding loops in other KH domains and contain conserved residues. KH domains are protein motifs that are involved in RNA binding and are often present in multiple copies. Here we demonstrate by coimmunoprecipitation studies that Sam68 self-associated and that cellular RNA was required for the association. Deletion studies demonstrated that the Sam68 KH domain loops 1 and 4 were required for self-association. The Sam68 interaction was also observed in Saccharomyces cerevisiae by the two-hybrid system. In situ chemical cross-linking studies in mammalian cells demonstrated that Sam68 oligomerized in vivo. These Sam68 complexes bound homopolymeric RNA and the SH3 domains of p59fyn and phospholipase Cgamma1 in vitro, demonstrating that Sam68 associates with RNA and signaling molecules as a multimer. The formation of the Sam68 complex was inhibited by p59fyn, suggesting that tyrosine phosphorylation regulates Sam68 oligomerization. Other Sam68 family members including Artemia salina GRP33, Caenorhabditis elegans GLD-1, and mouse Qk1 also oligomerized. In addition, Sam68, GRP33, GLD-1, and Qk1 associated with other KH domain proteins such as Bicaudal C. These observations indicate that the single KH domain found in the Sam68 family, in addition to mediating protein-RNA interactions, mediates protein-protein interactions.     

The STAR RNA binding proteins GLD-1, QKI, SAM68 and SLM-2 bind bipartite RNA motifs

Background  

SAM68, SAM68-like mammalian protein 1 (SLM-1) and 2 (SLM-2) are members of the K homology (KH) and STAR (signal transduction activator of RNA metabolism) protein family. The function of these RNA binding proteins has been difficult to elucidate mainly because of lack of genetic data providing insights about their physiological RNA targets. In comparison, genetic studies in mice and C. elegans have provided evidence as to the physiological mRNA targets of QUAKING and GLD-1 proteins, two other members of the STAR protein family. The GLD-1 binding site is defined as a hexanucleotide sequence (NACUCA) that is found in many, but not all, physiological GLD-1 mRNA targets. Previously by using Systematic Evolution of Ligands by EXponential enrichment (SELEX), we defined the QUAKING binding site as a hexanucleotide sequence with an additional half-site (UAAY). This sequence was identified in QKI mRNA targets including the mRNAs for myelin basic proteins.     

Inhibition of human immunodeficiency virus type 1 replication is enhanced by a combination of transdominant Tat and Rev proteins.

Mutation of either of two critical human immunodeficiency virus type 1 (HIV-1) regulatory proteins, Tat and Rev, results in marked defects in viral replication. Thus, inhibition of the function of one or both of these proteins can significantly inhibit viral growth. In the present study, we constructed a novel transdominant Tat mutant protein and compared its efficiency in inhibiting HIV-1 replication with that of transdominant mutant Rev M10 when these proteins were stably expressed either alone or in combination in T-lymphocyte cell lines. The transdominant Tat mutant protein alone resulted in a modest inhibition of HIV replication, but it was able to enhance the ability of the M10 Rev mutant protein to inhibit HIV-1 replication. These results suggest a possible synergistic effect of these transdominant mutant proteins in inhibiting HIV-1 replication.     

A new RNA element located in the coding region of a murine endogenous retrovirus can functionally replace the Rev/Rev-responsive element system in human immunodeficiency virus type 1 Gag expression

Nuclear export of incompletely spliced RNAs is a prerequisite for retroviral replication. Complex retroviruses like human immunodeficiency virus (HIV) encode a viral transport factor (Rev), which binds to its target sequence on the RNA genome and directs it into the Crm-1-mediated export pathway. Other retroviruses, like Mason-Pfizer monkey virus, contain cis-acting constitutive RNA transport elements (CTE) which achieve nuclear export of intron-containing RNA via cellular transport factors. Here, we describe the identification and characterization of a novel cis-acting orientation-dependent RNA expression element in the coding region of the murine intracisternal A-type particle (IAP) MIA14. This IAP expression element (IAPE) can functionally replace the Rev system in the expression of HIV-1 Gag proteins but functions independently of Crm-1. The presence of this element is needed for the expression of the IAP Gag proteins, indicating its biological significance. The IAPE can be functionally replaced by placing a CTE on the MIA14 RNA, further supporting its role in mRNA export. Northern blot analysis revealed that total RNA, as well as cytoplasmic RNA, was increased when the element was present. The element was mapped to a predicted stem-loop structure in the 3' part of the pol open reading frame. There was no overall homology between the IAPE and the CTE, but there was complete sequence identity between short putative single-stranded loops. Deletion of these loops from the IAPE severely reduced Rev-independent Gag expression.