The Shuttling SR Protein 9G8 Plays a Role in Translation of Unspliced mRNA Containing a Constitutive Transport Element

Abstract

The splicing regulatory SR protein, 9G8, has recently been proposed to function in mRNA export in conjunction with the export protein, Tap/NXF1. Tap interacts directly with the Mason-Pfizer monkey virus constitutive transport element (CTE), an element that enables export of unspliced, intron-containing mRNA. Based on our previous finding that Tap can promote polysome association and translation of CTE-RNA, we investigated the effect of 9G8 on cytoplasmic RNA fate. 9G8 was shown to enhance expression of unspliced RNA containing either the Mason-Pfizer monkey virus-CTE or the recently discovered Tap-CTE. 9G8 also enhanced polyribosome association of unspliced RNA containing a CTE. Hyperphosphorylated 9G8 was present in monosomes and small polyribosomes, whereas soluble fractions contained only hypophosphorylated protein.Our results are consistent with a model in which hypophosphorylated SR proteins remain stably associated with messenger ribonucleoprotein (mRNP) complexes during export and are released during translation initiation concomitant with increased phosphorylation. These results provide further evidence for crucial links between RNA splicing, export and translation.The serine/arginine (SR) 3 proteins are a family of RNA binding proteins with well recognized roles in splicing regulation (1-3). These proteins all have C-terminal arginine-and serinerich (RS) domains that can be phosphorylated and dephosphorylated on multiple serines by cellular kinases and phosphatases (2). The phosphorylation state is believed to play an important role in functional regulation (2, 4 -6).The effects of SR proteins on splicing have been extensively studied. Based to a large extent on in vitro studies, it has been well established that SR proteins, in general, bind to cis-acting regulatory sequences in many RNAs and promote splicing (7,8). Although individual proteins can have specific effects, SR proteins often appear to display overlapping and redundant functions (2,8).The SR proteins were initially thought to have solely nuclear functions, but it was subsequently shown that several of these proteins shuttle between the nucleus and the cytoplasm (9). The functional importance of this remained unclear until studies by Steitz and colleagues demonstrated a potential role for two of the shuttling SR proteins (SRp20 and 9G8) in mRNA export (10 -12). Another study reported the presence of SR proteins in polyribosomes, suggesting a potential role in translational regulation (13). Specifically, it was reported that the SF2/ASF protein was able to promote translation of mRNAs from reporter constructs containing SR protein binding sites. Several recent studies have now provided additional support of a role for SR proteins in translation (14,15).In the case of most mammalian genes, the primary RNA transcript contains multiple introns that must be removed by splicing before the mRNA can exit the nucleus (16 -18). Although it is not clear what restricts mRNA from export before splicing has been completed, the shuttling protein, Tap/NX...

Mutually exclusive interactions drive handover of mRNA from export adaptors to TAP

“…However, other proteins may be limiting in this situation, and the stable recruitment of TAP to mRNA may require factors such as U2AF or the Cap-binding complex. Interestingly, 9G8 has recently been shown to enhance expression of genes containing a constitutive transport element (CTE) (31), which may arise through enhanced TAP-CTE interaction. Clearly, further experiments are required to resolve this issue and further establish whether loss of an export adaptor…”

Serine/arginine-rich splicing factor 7 regulates p21-dependent growth arrest in colon cancer cells

“…SRSF7 recognizes CTEs of virus mRNAs through its RRM domain, and contributes to translational activation of these mRNAs as well as mRNA export of them (20,36). A subset of RNA-binding proteins, SRSF3 and RBM4, regulate translational activity of their targeted mRNAs through binding to 5'UTR of them (22,27).…”

“…SRSF7 shuttles constantly between the nucleus and cytoplasm (10), and contribute to the export of certain viral and cellular mRNAs through directly binding to transporter 1 (TAP)/nuclear RNA export factor 1 (NXF1), an essential mRNA export factor (16,20,21,32). In addition, overexpression of SRSF7 enhanced the association of polyribosome with a structured RNA sequence, termed the constitutive transport element (CTE), which recruits TAP/NXF1 (36).…”

The Tpr protein regulates export of mRNAs with retained introns that traffic through the Nxf1 pathway

“…shown that expression and translation of the GagPol produced from this reporter can be monitored by measuring the accumulation of HIV p24 in the supernatant of the transfected cells (Smith et al 1990;Bray et al 1994;Guzik et al 2001;Coyle et al 2003;Jin et al 2003;Bor et al 2006;Li et al 2006;Swartz et al 2007). Such expression is absolutely dependent on the presence of a cis-acting element, either a CTE or an RRE.…”