Identification of the sequence determinants mediating the nucleo-cytoplasmic shuttling of TIAR and TIA-1 RNA-binding proteins

Abstract

TIAR and TIA-1 are two closely related RNA-binding proteins which possess three RNA recognition motifs (RRMs) followed by an auxiliary region. These proteins are involved in several mechanisms of RNA metabolism, including alternative hnRNA splicing and regulation of mRNA translation. Here we characterize the subcellular localization of these proteins in somatic cells. We demonstrate that TIAR and TIA-1 continuously shuttle between the cytoplasm and the nucleus and belong to the class of RNA-binding proteins whose nuclear import is transcription-dependent. We identified RRM2 and the first half of the auxiliary region as important determinants for TIAR and TIA-1 nuclear accumulation. In contrast, the nuclear export of TIAR and TIA-1 is mediated by RRM3. Both RRMs contribute to TIAR and TIA-1 nuclear accumulation or export by their RNA-binding capacity. Indeed, whereas mutations of the highly conserved RNP2 or RNP1 peptides in RRM2 redistribute TIAR to the cytoplasm, similar modifications in RRM3 abolish TIAR nuclear export. Moreover, TIAR and TIA-1 nuclear accumulation is a Ran-GTP-dependent pathway, in contrast to its nuclear export which is unaffected by Ran-GTP depletion and which is independent of the major CRM1-exporting pathway. This study demonstrates the importance of TIAR and TIA-1 RNA-binding domains for their subcellular localization and provides the first evidence for distinct functions of TIAR and TIA-1 RRMs.

An RNA Recognition Motif Mediates the Nucleocytoplasmic Transport of a Trypanosome RNA-binding Protein

“…TIA-1 and TIAR are nuclear proteins composed of three RRMs, of which RRM2 plus its COOH-terminal auxiliary domain are needed for nuclear accumulation (23). Similarly to TcUBP1, the disruption of TIAR RNA binding activity by point mutations in either RNP2 or RNP1 impairs nuclear accumulation (23).…”

“…Non-classical NLS were described (20), including different RRMs in PABP1 (21,22), TIA-1, and TIAR (23). Notably, TIA-1 and TIAR nuclear accumulation is dependent on active transcription (23), as it is the case also for heterogeneous nuclear ribonucleoprotein A1 (24), HuR (25), and many other RBPs (see "Discussion"). Once in the nucleus, some proteins also shuttle back to the cytoplasm by virtue of the specific recognition of a nuclear export signal by the respective exportin.…”

“…These NLS are recognized in the cytoplasm by importin ␣ and ␤, which in the presence of Ran-GDP, NTF2, and GTP are translocated across the NPC (19). Non-classical NLS were described (20), including different RRMs in PABP1 (21,22), TIA-1, and TIAR (23). Notably, TIA-1 and TIAR nuclear accumulation is dependent on active transcription (23), as it is the case also for heterogeneous nuclear ribonucleoprotein A1 (24), HuR (25), and many other RBPs (see "Discussion").…”

Nucleocytoplasmic Traffic of CPEB1 and Accumulation in Crm1 Nucleolar Bodies

“…NES1* (L99L102L104AAA) and NES2* (I202L205I207AAA) point mutations were created in the CPEB1-short expression vector, whereas RRM1* (F314A) and RRM2* (F433A) point mutations were created in the CPEB1-long expression vector, using the QuickChange XL site-directed mutagenesis kit (Stratagene, La Jolla, CA). GFP-tagged polypyrimidine tract binding protein (PTB) and TIA1 (Zhang et al, 2005) were a kind gift of David Spector (CSH Laboratories, Cold Spring Harbor, NY) and Veronique Kruys (Institute of Biology and Molecular Medicine, Charleroi-Gosselies, Belgium), respectively.…”

“…For instance, Rck/p54 which is essential for the repression of maternal mRNA in the cytoplasm in combination with CPEB1 (Minshall et al, 2001), is both nuclear and cytoplasmic in early Xenopus oocytes (Smillie and Sommerville, 2002). Such a dual localization is observed for various factors, including CUG-BP/EDEN-BP (Fujimura et al, 2008), TIA1 (Lopez de Silanes et al, 2005;Zhang et al, 2005), TIAR (Mazan-Mamczarz et al, 2006), FMRP, FXR1, and FXR2 (Eberhart et al, 1996;Tamanini et al, 1999). In some cases, for This article was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E08 -09 -0904) on October 15, 2008. instance CUG-BP/EDEN-BP, TIA1, and TIAR, it reflects a dual function of the protein in both translation and splicing (Philips et al, 1998;Le Guiner et al, 2001).…”