www.landesbioscience.com RNA Biology 339

RNA Biology 7:3, 339-344; May/June 2010; © 2010 Landes Bioscience

PoiNt of View PoiNt-of-View

Key words: RNA recognition motif, RNA-binding protein, ribonucleoprotein complex, nuclear localization signal, mRNA export, Trypanosoma

Abbreviations: PABPC1, poly(A) bind-ing protein 1; RBP, RNA-binding pro-teins; RRM, RNA recognition motif; NLS, nuclear localization signal; NPC, nuclear pore complexes; LA-NLS, LA protein heterologous NLS; UTR, untranslated region; mRNP, messenger ribonucleoprotein

Submitted: 02/22/10

Accepted: 04/14/10

Previously published online: www.landesbioscience.com/journals/rnabiology/article/12087

*Correspondence to: Alejandro Cassola; Email: acassola@iib.unsam.edu.ar

In eukaryotic cells, a regulated import and export of factors is required to

fulfill the requirements of precise gene expression. Post-transcriptional regula-tion of gene expression has proven to pro-vide ubiquitous control, as well as a quick response to environmental changes when required. RNA-binding proteins (RBP) are involved in the several steps at which mRNA biogenesis, stability, translation and decay is exerted. The most charac-terized RBPs contain single or multiple copies of an RNA Recognition Motif (RRM). Here, we concentrate on RRMs mediating protein nuclear import by vir-tue of its ability to interact with proteins, besides interacting with nucleic acids. The consensus on how RRM-protein interactions take place is non-existent, and so is the involvement of the RRM as a nuclear localization signal (NLS). Within the cases examined, the single RRM from a trypanosome RBP behaves as a structural NLS, alternating nuclear import and RNA-binding.

Introduction

The nuclear envelope is a distinctive char-acteristic of eukaryotic cells. All nucleo-cytoplasmic transport occurs through nuclear pore complexes (NPC), which allow the passive diffusion of molecules below 40 kDa.1 Active transport of mol-ecules is mediated by soluble factors from the karyopherin-β/importin-β protein family, namely importins and exportins.1 Most karyopherins-β interact directly with the transported protein, although they can also interact through an adapter protein, being importin-α the most

studied example.2 Nuclear import of pro-teins was first described for nucleoplasmin and SV40 large T antigen. Both proteins have conserved NLS frequently found in other proteins. These signals, known as classical or basic NLS, are necessary and sufficient for transport, and are commonly recognized by importin-α. Classical NLS are typically short, and composed by one (monopartite) or two stretches (bipar-tite) of charged amino acids.2 There are other types of NLS composed by larger domains with a reduced number of charged amino acids, where the spatial structure of the domain likely determines the interaction with the import receptor or adapter. Examples of these NLS are the M9 of hnRNP A1,3 the HNS in HuR,4 the RRM of TcUBP1,5 the RRM2 plus the first half of the auxiliary domain in TIA-1 and TIAR,6 a fragment spanning the RRM in Saccharomyces cerevisiae LA protein, Lhp1p,7 the first two RRMs in the cytoplasmic Poly(A) binding protein 1 (PABPC1),8 and a fragment encompass-ing the COOH-terminus of RRM3 and the amino-terminus of RRM4 in yeast PABP1, Pab1p.9 Although all these exam-ples correspond to RBPs, unconventional nuclear localization motifs can be found in proteins with diverse function.

In this work we focus on RRM-type RBPs that have the ability to enter the cell nucleus. In these proteins, one or more RRMs are recognized partially or com-pletely as NLS, that are different to clas-sical NLS except for one case. As a model where solely an intact and functional RRM mediates nuclear import, we will describe the case of the TcUBP1, a single RRM RBP present in unicellular trypanosomes. The

RNA recognition motifs involved in nuclear import of RNA-binding proteins

Alejandro Cassola,* Griselda Noé and Alberto C. FraschInstituto de Investigaciones Biotecnológicas—Instituto Tecnológico Chascomús; UNSAM-CONICET; Buenos Aires, Argentina

340 RNA Biology Volume 7 issue 3

protein-protein interactions (reviewed in ref. 12), and in protein nuclear import. One of the most representative examples of an RRM involved in nuclear import is provided by the single RRM of TcUBP1 from Trypanosoma cruzi.5 In this early divergent protozoan parasite, the causative agent of Chagas disease, regulation of gene expression is mostly post-transcriptional.15 In TcUBP1, the 92 residue-long RRM (aa. 35–126) is the minimal portion of the protein allowing nuclear import5 (Fig. 2). Another versatile RRM is the one in Lhp1p, the ortholog of the LA pro-tein from S. cerevisiae involved in biogen-esis and processing of noncoding RNAs.16 In this nuclear protein, the NLS has been determined as a 113-amino acid region (aa. 112–224) that includes the single RRM (aa. 124–209)7 (Fig. 2). The exact boundaries of the Lhp1p NLS have not been clearly defined, and could probably be smaller than described.

In the group of proteins containing multiple RRMs, the nuclear RBPs TIA-1 and TIAR have shown to require RRM2 and the first half of the auxiliary region located at the COOH-terminus for proper nuclear import and localization6 (Fig. 2). These are nucleocytoplasmic shuttling proteins involved in pre-mRNA splicing in the nucleus,17,18 and regulation of mRNA translation19-21 and Stress Granules forma-tion22 in the cytoplasm. Another interest-ing case where more than one RRM is involved in nuclear import is the case of PABPC1, where the first two RRMs are

(RNP1 position 1) (reviewed in ref. 10). Recent structural studies, however, have proven a role for the loops connecting β-strands and α-helices in nucleic acid recognition, resulting in enlarged RNA-binding surfaces (reviewed in ref. 12).

RRM-type proteins are involved in post-transcriptional regulation of gene expression, from RNA processing and export in the nucleus, to mRNA trans-lation, stability and decay in the cyto-plasm.12 RNA trans-acting factors are directed towards specific RNAs using the RRM as an RNA-binding platform. Binding is achieved primarily over single-stranded sequences, which are usually taking part of structured RNAs.13 Given that the residues involved in direct inter-actions with RNA are conserved between RRMs from yeast to humans, affinity of different RRMs is achieved through the usage of external β4 and β2 strands, or β1α1, β2β3 or α2β4 loops. Specificity of an RBP towards a target RNA might be determined by multiple RRMs in the same protein, since a single RRM can only recognize a small number of nucleotides. Thus, more than one RRM can form a larger binding surface that allows the binding of a longer RNA sequence with high affinity.10,14

RRMs Involved in Protein Nuclear Import

Over the past years, RRMs have gained attention due to their involvement in

relevance of RRM-mediated nuclear pro-tein transport will be discussed in the con-text of the structural recognition of RNA and proteins directed by RRMs, both in the nucleus and the cytoplasm.

The RNA Recognition Motif: Structure and RNA Recognition

The RRM is the most abundant RNA-binding module, being present in the protein product of around 1% of human genes.10 This domain is composed of approximately 90 amino acids that adopt a βαββαβ topology (Fig. 1). The fold forms a four-stranded antiparallel β-sheet with the two α-helices packed against it11 (Fig. 1). The surface of the β-sheet usu-ally mediates recognition of the RNA as an open platform, where the number of bound nucleotides ranges from two to eight.12 Within the β-sheet, the β1 and β3 central strands harbor the RNP2 and RNP1 motifs, respectively (Fig. 1). The highly conserved RNP1 consen-sus sequence is an octapeptide defined as K/R-G-F/Y-G/A-F/Y-V/I/L-X-F/Y, while the RNP2 sequence is a less con-served stretch of six residues defined as I/V/L-F/Y-I/V/L-X-N-L.10 Typically, there are four residues that contribute to RNA binding, which are positions 1, 3 and 5 in RNP1, and position 2 in RNP2. Charged and aromatic side chains of these residues are exposed to the solvent, and interact directly with the RNA through stacking interactions and one salt bridge

Figure 1. Structure of an RRM. (A) Ribbon diagram of a typical RRM from TcUBP1 based on NMR spectroscopy structural data (Protein Data Bank acces-sion number 1U6f).36 in this RRM, a β-hairpin motif can be found between α2 and β6. β5 and β6 strands are shown together. (B) Primary and second-ary structure of TcUBP1 RRM comprised between amino acids 35 to 126. β strands are shown as white boxes, and α helices are shown as black spirals. the positions or the conserved RNP2 and RNP1 motifs are indicated as black boxes in the sequence.

www.landesbioscience.com RNA Biology 341

TcUBP1 RRM: One for All

TcUBP1 is a protein that is involved in mRNA stabilization in the cytoplasm.30 Also, it plays a central role in the protec-tion of cytoplasmic transcripts in large P body-like structures termed mRNA granules that are formed under starva-tion conditions.31 Recently, we found that nuclear TcUBP1 can also be detected in small amounts given the nucleocyto-plasmic shuttling nature of this protein. TcUBP1, and the highly related RBP TcUBP2, gradually accumulate in the nucleus of arsenite-stressed parasites5 (Fig. 3A). The minimal portion of the protein retaining the ability to accumulate in the nucleus was narrowed to the 92-residue RRM (TcUBP1 ∆N∆QG2 in Fig. 3B). This region of the protein is sufficient for proper normal localization,5 RNA-binding,32 as well as recruitment to mRNA

compared with the ones described above, and is rather similar to classical NLS. Given its arginine and lysine content, this NLS is restricted to the COOH-terminus of the RRM, where interactions with RNA are not taking place.

In all of the above described proteins there are also domains involved in nuclear export, such as the RRM3 in TIA-1 and TIAR,6 residues 296 to 317 in RRM4 of PABPC1,29 RRM1 in Pab1p,9 the first two RRMs and part of the divergent domain in ETR-3.27 The single RRM of Lhp1p was also suggested to be involved in export to the cytoplasm,7 although there is no experimental data support-ing this notion. In the case of TcUBP1, the complete and functional RRM is required not only for nuclear import, but also mediates export to the cytoplasm.5 It is the versatility of this trypanosomatid RRM what will be discussed in the fol-lowing sections.

recognized as an NLS8 (Fig. 2). PABPC1 is a nucleocytoplasmic shuttling protein,8 which has been proposed to bind polyade-nylated transcripts in the nucleus.23 In the cytoplasm, it is involved in mRNA trans-lation, stability and decay (reviewed in ref. 24).

The yeast ortholog of PABPC1, Pab1p, has a completely different NLS. It is located between amino acids 281–338, superpos-ing with the COOH-terminus of RRM3 and the amino-terminus of RRM4,9 (Fig. 2). Pab1p shuttles rapidly between the nucleus and the cytoplasm,9 and has been proposed to play a role in mRNA biogenesis, stability and translation (reviewed in refs. 25 and 26). Another NLS is found inside the third RRM of ETR-3,27 (Fig. 2), a nucleocytoplasmic RBP proposed to regulate alternative splicing, RNA editing, mRNA stabil-ity and translation events (reviewed in ref. 28). This NLS is relatively short

Figure 2. RRMs involved in nuclear import. A domain scheme for each of the proteins is shown. As indicated, the position of the RRMs were numbered according to PS50102 motif profile in PRoSite,37 except for TcUBP1,5 tiA-1 and tiAR.6 inside each RRM, the position of the RNP2 and RNP1 motifs are shown to scale by grey and black bars, respectively. Additional motifs are indicated as black boxes as follows: Q, glutamine rich; G, glycine rich; LA, LA motif; Aux., auxiliary domain; PABC, PABP conserved CooH-terminal domain. the positions of the identified NLS are shown according to experimental evidence. in the case of PABPC1 and Pab1p only the relevant parts of the proteins are shown.

342 RNA Biology Volume 7 issue 3

motif in TcUBP1 RRM (mTcUBP1 ∆N ∆QG2) abrogates nuclear import, as well as RNA binding, normal localization, and association with mRNA granules5 (Fig. 3B). As a whole, the experimental evidence argues for a perturbation of the

β6 strands at the COOH-terminus renders mutant RRMs that distribute through-out the entire cell in all tested conditions (Fig. 3B). Most notably, mutating sol-vent exposed amino acids in positions 1 (R84), 3 (Y86) and 5 (F88) of the RNP1

granules.31 Although this single RRM is small enough to passively diffuse through nuclear pore complexes, it is actively trans-ported into the nucleus. Deleting either the β1 strand (containing the RNP2 motif) at the amino-terminus, or β5 and

Figure 3. for figure legend, see page 343.

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export, a blockage in the export of mRNP complexes is most probably the cause of the accumulation of this protein in the nucleus under stress, a phenomenon that would otherwise be unnoticed.

Future Challenges

The function of RRMs in protein nuclear import relies on the ability of these domains to interact not only with nucleic acids but also with proteins.12 Over the past years, the determination of the structure of cer-tain RRMs in complex with their RNA or protein interacting partners has led to the understanding of how versatile this highly conserved domain can be. The structural analysis of RRM-protein complexes have shown that there is not a general mecha-nism underlying these interactions.10 Up to now, only Lhp1p and Pab1p have been honored with the identification of an inter-acting importin, which in both cases con-sisted of Sxm1/Kap108.7,9 Given the high frequency of RRMs in eukaryotic gene products, it is not surprising to find much more examples of cryptic nuclear localiza-tion motifs involved in nuclear import of post-transcriptional modulators. Of much interest would be to obtain the resolved structures of cargoes-transporters in order to determine the domain structures involved in nuclear import. The knowledge of the chemical nature of these interactions would allow interfering with a limiting step in the functionality of shuttling RBPs.

Acknowledgements

This work was supported by grants from the Agencia Nacional de Promocion Científica y Tecnológica (ANPCyT, Argentina) (to A.C.F.), an International

signature RNA motif with a stem-loop structure, called UBP1m (Fig. 3C), fre-quently found within the 3' untranslated region (UTR) of mRNAs. Interestingly, in some target-transcripts the surrounding sequence of UBP1m harbors overlapping RNA motifs for other RBPs,34 suggesting that it can adopt more than one structure depending on the presence of TcUBP1 or other competing factors. In this context, TcUBP1 recognition of UBP1m in target mRNAs could be taking place either on nuclear monocistrons or polycistrons. In fact, TcUBP1 can co-localize with mRNA targets and bulk poly(A)+ mRNA in the nucleus under stress conditions.5 In nor-mal conditions, the nuclear availability of TcUBP1 might determine the structure of the 3'UTR elements found in newly synthesized transcripts, contributing to maintain a specific RNA folding and aid-ing to thermodynamic stability.35 The prevalence of a determined RNA struc-ture among others, would drive messen-ger ribonucleoprotein (mRNP) complexes composition and fate before reaching the cytoplasm (Fig. 3D). Once exported to the cytoplasm, TcUBP1-containg mRNP complexes are remodeled in order to spec-ify the subcellular localization, translation and stability of the mRNA30,31 (and our unpublished results). These events are probably determined by the precise com-position of the nuclear assembled mRNP complex. Once liberated from a cyto-plasmic mRNP complex, TcUBP1 and TcUBP2 are recycled from the cytoplasm to the nucleus, using the RRM as a struc-tural moiety for the interaction with a yet unidentified importin (Fig. 3D).

Given the dependence of RNA-binding for proper TcUBP1 nuclear

three-dimensional structure of TcUBP1 RRM made by these deletion and point mutations, which blocks the recognition by an import receptor or adapter.

Once in the nuclear compartment, TcUBP1 RRM can be efficiently exported to the cytoplasm if arsenite stress is termi-nated, and normal conditions are restored. In fact, TcUBP1 is continuously exported from the nucleus by virtue of the RRM. This domain is so efficiently exported from the nucleus that it can overcome nuclear localization imposed by the heterologous LA protein NLS (LA-NLS). Consequently, introduction of the RNP1 mutations, which prevent binding to RNA, block the normal nuclear export of TcUBP1 RRM fused to the LA-NLS.5 Altogether, the evi-dences gathered here point out the multi-plicity of functions that this single RRM confers to this RBP, and grants speculation about TcUBP1 being exported from the nucleus as a cargo on mRNA.

Relevance of TcUBP1 Nuclear Import

In the trypanosome nucleus, pre-mRNAs are synthesized as long polycistronic units that are processed into monocistrons by coupled events of trans-splicing and poly-adenylation.33 Hence, regulation of gene expression is mainly governed post-tran-scriptionally in the cytoplasm by RBPs such as TcUBP1.15 This protein interacts in the cytoplasm with TcPABP1,32 and can also be detected in association with polysomes (De Gaudenzi and Frasch AC, unpublished results).

Recently, we have identified the mRNA targets of TcUBP1.34 The associa-tion to these transcripts is mediated by a

Figure 3 (See opposite page). RRM-dependent TcUBP1 behavior. (A) TcUBP1 nuclear accumulation dynamics. TcUBP1-GfP localization in T. cruzi transfected parasites incubated with 2 mM arsenite for the indicated time periods. DAPi staining, shown in red for better contrast, reveals the posi-tion of nuclear (N) and mitochondrial (k, kinetoplast) DNA. Scale bar: 5 µm. (B) RRM-mediated nuclear import. A brief description of the localization of TcUBP1 and mutant constructs are shown. Sequence coverage is indicated between parenthesis and point mutations, indicated by an asterisk (*), between brackets (parental numeration). Normal refers to the localization of constructs in physiologically cultured parasites, and Arsenite refers to the localization of constructs in parasites incubated with 2 mM arsenite for 4 hours. C, cytoplasmic; N, nuclear; C>N, more cytoplasmic than nuclear; C = N, even in cytoplasm and nucleus. (C) TcUBP1 target transcripts harbor an UBP1m signature RNA motif within the 3'UtR. the sequence and the stem-loop structure of the UBP1m motif are shown. RNA-binding assays were used to demonstrate that this element is involved in the interaction of TcUBP1 with target mRNAs. A non-target biotinylated RNA and the same transcript containing UBP1m motif were incubated with TcUBP1. RNA-protein complexes were recovered using streptavidin-conjugated beads and the presence of TcUBP1 was detected by western blotting. (D) A model for TcUBP1 and TcUBP2 nucleocytoplasmic dynamics. in the nucleus, the local availability of TcUBP1 allows binding to UBP1m in the 3'UtR of target transcripts. this might stabilize an RNA secondary structure that would define the identity of the newly formed mRNP, together with TcUBP2. this mRNP is rapidly ex-ported to the cytoplasm, where it can be translated, stored into mRNA granules, or degraded, according to the requirements of the cell. once TcUBP1 and TcUBP2 are released from the mRNP, the proteins are imported back into the nucleus by an unidentified receptor or adapter that specifically recognizes the RRM. SL, spliced leader; imp, importin.

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Research Scholars Grant from the Howard Hughes Medical Institute (to A.C.F.) and the National Institutes of Health grant AI0600645 (to A.C.F.). The project described was supported by Grant Number D43TW007888 from the Fogarty International Center. The content is solely the responsibility of the authors and does not necessarily represent the official views of the Fogarty International Center or the National Institutes of Health. A.C. and G.N. are research fellows, and A.C.F. is a researcher from the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Argentina.

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