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FEBS Letters 582 (2008) 2345–2351

Minireview Spliceosomal immunophilins

Annia Mesa1, Jason A. Somarelli1, Rene J. Herrera* Florida International University, Department of Biological Sciences, University Park, 11200 SW 8th Street, OE 304, Miami, FL 33199, United States

Received 14 April 2008; revised 16 May 2008; accepted 2 June 2008

Available online 9 June 2008

Edited by Daniela Ruffell

pendent reaction, creating the E complex [6] (Fig. 1). The U1 Abstract The spliceosome is a dynamic, macromolecular com- plex, which removes non-protein-coding introns from pre-mRNA snRNP promotes the association of U2 with the branch point to form mature mRNA in a process known as splicing. This ribo- intronic sequence in an ATP-dependent manner (the A com- nucleoprotein assembly is comprised of five uridine-rich small nu- plex) [7], establishing a favorable conformation for the first clear RNAs (snRNAs) as well as over 300 proteins. In humans, trans-esterification reaction [8,9] (Fig. 1). Next, a tri-snRNP several of the known proteinaceous splicing factors are members composed of the U4/U6 di-snRNP and the U5 snRNP interact of the immunophilin superfamily. Immunophilins are peptidyl- with the U2 snRNP to produce the B1 complex [10] (Fig. 1). prolyl cis–trans isomerases that catalyze the conversion of pro- The first trans-esterification reaction takes place in the B2 com- teins from cis to trans at Xaa-Pro bonds. Our review of the data plex, creating a free 50 exon and a lariat intron/30 exon [11] indicates that some members of this protein family are activators (Fig. 1). The C1 spliceosome is responsible for the second of spliceosomal proteins by way of folding and transport. trans-esterification, where the 30 end of the intron is truncated, Ó 2008 Federation of European Biochemical Societies. Pub- 0 lished by Elsevier B.V. All rights reserved. separating the intron from the 3 exon. At this time, the U5 snRNP holds the two exons in close proximity [9]. Finally, Keywords: Cyclophilin; FKBP; Spliceosome; Cyclosporin; C2 is formed by two ligated exons, an independent lariat FK506; Small nuclear RNAs intron as well as the U2, U5 and U6 snRNPs [12] (Fig. 1). Subsequent to production of the ligated exons, the U2/ U6.U5 tri-snRNP complex disassembles into individual snRNPs and are recycled in preparation for the next round of splicing [13] (Fig. 1). 1. Introduction In mammalian systems, over 300 proteins participate in splicing, making the spliceosome one of the largest cellular Most of the transcripts produced in the eukaryotic cell are complexes discovered thus far [14,15]. Many of these proteins comprised of both protein-coding exons and non-protein-cod- have been previously identified and participate in a variety of ing introns. Following transcription, the introns are removed additional cellular processes including transcription regula- and the exons ligated together by a dynamic, high molecular tion, intra- and inter-cellular transport, apoptosis, kinase weight RNA–protein complex known as the spliceosome [1]. activity and protein folding (Fig. 2). Proteomics analyses of This macromolecular complex is composed of five uridine-rich the human spliceosome identified several splicing factors that small nuclear RNAs (snRNAs) designated U1, U2, U4, U5 are members of the immunophilin family [14,16]. The inmuno- and U6 each bound to a core set of snRNA-specific proteins philins represent a diverse group of proteins that are defined by as well as numerous other proteinaceous splicing factors which their peptidyl-prolyl cis–trans isomerase (PPIase) activity and form small nuclear ribonucleoprotein particles (SnRNPs) [2,3]. are known to bind immunosuppressive drugs [17]. They cata- Some of these proteins are an essential part of the spliceosome lyze the conversion of proteins from cis to trans at Xaa-Pro while others perform auxiliary roles in the processing of tissue- bonds, which is often a rate-limiting step in protein folding and/or developmental stage-specific transcripts. and activity [18]. The first of the immunophilins was isolated The snRNAs form distinct stem-loop structures that facili- in 1984 from bovine thymocytes as a molecule with high affin- tate snRNA-protein and snRNA pre-mRNA interactions [4]. ity for the immunosuppressive drug cyclosporin A (CsA) [19]. The mechanism of splicing involves a cascade of assembly, It was subsequently discovered that this protein also exhibits trans-esterification and disassembly reactions to produce two PPIase activity [20]. The immunophilin superfamily is classified ligated exons and an excised intron, which is subsequently de- into three major subfamilies, including the cyclosporin A graded [5] (see Fig. 1 for a diagrammatic representation of the (CsA) binding proteins (cyclophilins or Cyps) [21], FK506 pre-mRNA splicing process). Following transcription of the binding proteins (FKBPs) [22] and parvulins [23]. In addition, pre-mRNA, the U1 small nuclear ribonucleoprotein particle other proteins bridge the two subfamilies by possession of both (snRNP) binds to the 50 exon–intron junction in an ATP inde- cyclophilin and FKBP domains, like the one identified in the protozoan, Toxoplasma gondii [24]. Immunophilins have been documented in every Domain of the tree of life including *Corresponding author. Fax: +1 305 349 1259. Archaea, Bacteria and Eukaryota and they are found in a E-mail address: herrerar@fiu.edu (R.J. Herrera). number of cellular compartments including the mitochondria, endoplasmic reticulum, Golgi, nucleus and cytoplasm [17,25]. 1These authors contributed equally to this work.

0014-5793/$34.00 Ó 2008 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2008.06.006 2346 A. Mesa et al. / FEBS Letters 582 (2008) 2345–2351

U1 E

E1 A E2 degrading intron

ATP AD P A U2

E1 A E2 A lariat intron

A

U4/U6

E1 E2 E1 ligated A I B1 exons A E2 ADP ATP U5 ADP ATP

E1 E2 E1 C2 A B2 E Exon A E2

ADP ADP ATP ATP U1 snRNP E1 A E2 U2 snRNP C1 U4 snRNP

U5 snRNP

U6 snRNP

Fig. 1. An overview of pre-mRNA splicing. Removal of non-protein coding introns from pre-mRNA involves a dynamic, high molecular weight complex known as the spliceosome. This nucleo-protein assemblage is comprised of five uridine rich small nuclear RNAs (snRNAs) as well as over 200 splicing proteins. All small nuclear ribonucleoprotein particles (snRNPs) (U1, U2, U4/U6 di-snRNP and U5) are labeled as they incorporate into the spliceosomal complex. E1 and E2 represent exons, separated by an intron (line) containing the branch point sequence (symbolized by an A). The names of each complex (E, A, B1, B2, C1, C2 and I) are designated in bold. ATP dependent reactions are indicated along the cycle. The degrading intron is symbolized by a dashed line.

Table 1 Immunophilins of the spliceosome Human immunophilin Putative yeast homologue Predicted size (kDa) Known functional domains Interacting proteins (Human/Yeast) PPIH, Snu-Cyp20, CYP3 (S. pombe) 19/19 Cyclophilin domain hPrp4, hPrp18 USA-Cyp, CypH

PPIG, SR-Cyp, – 89/– SR domain, Cyclophilin domain, Clk/Sty, RNA polymerase II CARS-Cyp, CypG Nopp140 related domain

PPIE, Cyp33, CypE – 33/– RRM, Cyclophilin domain hSAP49

PPIL1 CYP1 (S. pombe) 20/18 Cyclophilin domain 45S snRNP, 35S U5 snRNP, SKIP, stathmin

PPIL2, Cyp60 Cyp60 (R. oryzae) 60/61 Ubox domain, Cyclophilin domain, CD147, eglin C Uncharacterized conserved region

PPIL3b, CypJ – 18/– Cyclophilin domain –

All immunophilins possess at least one PPIase domain, philins interact directly with splicing factors [29] and make which modulates most of their cellular functions including up part of the spliceosome. Some also bind to the carboxyl ter- folding and chaperone activities [17,24]. A number of studies minal domain (CTD) of RNA polymerase II (RNA pol II) have detected the presence of immunophilins in the nucleus. [26]. The involvement of immunophilins in these processes These include PPIG [26], PPIE [27], PPIL1 [28], PPIH [29], seems to be an ancient and universal trait due to their presence PPIL2 [16] and PPIL3a/PPIL3b [16]. These nuclear immuno- in the splicing and transcriptional machinery in a multitude of A. Mesa et al. / FEBS Letters 582 (2008) 2345–2351 2347

A Lariat intron Protein kinase

Microtubule Assembly RNA polymerase II Clk/Sty Kinase Phosphate group SAP145

RNA Pol II T Stathmin Microtubulin subunit

Golgi vessicle T T T T CD 147 receptor

T T T T T T Transcription SKIP

Prp3 Prp4

Prp18 Nucleus Stathmin

HsCyp89 Cytosol HsCyp33

A SAP145 HsCyp20

I HsCyp60

A HsCyp19 B1 A HsCyp18 C2 Prp4 Transport of CD147 from Golgi U1 snRNP A B2 Prp3 Exon

A SKIP U2 snRNP C1 U4 snRNP Prp18 A U5 snRNP

U6 snRNP

Prp4 Prp3

Fig. 2. Potential functions of spliceosomal immunophilins in the mammalian cell. Based on the available data, the hypothesized entry and exit points for each immunophilin into the spliceosome are delineated by arrows. In the cases of PPIL3b and PPIL2, dashed arrows are employed to indicate unknown stages at which these proteins are incorporated into the spliceosome. In addition to their putative roles in splicing, these immunophilins may modulate transcription regulation, microtubule assembly, folding and intra- and inter-cellular transport of proteins from the endoplasmic reticulum and Golgi to locations like the cell membrane. taxa. Furthermore, several studies based on proteomics have number of spliceosomal immunophilins have been identified identified cyclophilins in spliceosomal complexes (reviewed in in several taxa, including matrin-Cyp in Rattus norvegicus [30,31]). Interestingly, each of the analyses differs in the num- [33] and AtCyp59 in Arabidopsis thaliana [34]. The overall ber and types of cyclophilins that are found in the spliceosome theme that emerges from our exploration of the literature is [31]. For example, Chen and coworkers [31] found six cyclo- that immunophilins are capable of catalyzing a number of philins that co-purify with chicken spliceosomes (PPIE, PPIH, transport and foldase reactions that possibly assist in the serologically defined colon cancer antigen 10 (SDCCAG10), assembly/disassembly dynamics of the spliceosome as well as PPIL1, PPIL2 and peptidyl prolyl isomerase domain and in its coupling to the transcription machinery. Furthermore, WD repeat containing 1 (PPWD1)). Rappsilber et al. [14] also it is feasible that the capacity of immunophilins to alter folding identified PPIL3b as part of the human spliceosome, as well as may provide accessibility and a mechanism for activating/inac- all of the cyclophilins detected by Chen et al. [31] with the tivating spliceosomal proteins. In this review, we focus on the exception of PPIH. Another analysis detected PPIE, CYP16, immunophilins of the human and yeast spliceosomes, as proto- PPIL1, PPIL2, PPIL3b and PPWD1 in human spliceosomes, typic examples of the potential roles that immunophilins play but not PPIH and SDCCAG10 [16]. The discrepancies among in splicing as well as in additional functions. the three studies may be related to species and/or tissue-specific diversity in spliceosomal components. There is precedence for 2. Spliceosomal immunophilins tissue-specific variation in cyclophilin expression [32]. The dif- ferences observed may also allude to the dynamic nature in 2.1. PPIH associates with the hPrp4 and hPrp18 splicing factors which these proteins associate with the spliceosome. PPIH, also known as Snu-Cyp20, USA-Cyp or CypH in hu- Given the constitutive and ubiquitous nature of RNA pro- mans, is a 19 kDa nuclear protein that contains a single PPIase cessing, the presence of immunophilins in the splicing machin- domain and interacts specifically with the splicing factors ery may be significant in modulating spliceosomal activity. A hPrp3, hPrp4 and hPrp18 [35–38] (Fig. 2, Table 1). Both hPrp3 2348 A. Mesa et al. / FEBS Letters 582 (2008) 2345–2351 and hPrp4 are human splicing factors that integrate into the RNA splicing through phosphorylation of splicing factors U4/U6 di-snRNP and are essential for the initial steps of the [43] (Fig. 2). PPIG was also identified as a nuclear matrix pro- splicing reaction [39]. The hPrp18 splicing factor is involved tein that assembles with splicing factors in nuclear speckles in the assembly of the U2/U6.U5 tri-snRNP and functions in [26], which are known to contain high concentrations of the second trans-esterification reaction [40]. Horowitz et al. RNA processing components [44]. Nestel et al. [32] identified [29] were able to elute complexes of [3H] CsA, His-tagged PPIG mRNAs in 13 human tissues and cell types, but could PPIH and hPrp4 as well as [3H] CsA, His-tagged PPIH and not detect expressed transcripts corresponding to PPIG in nat- hPrp18 from affinity columns. This finding suggests that inter- ural killer (NK) cells, where the expression of the NK-TR1 actions between PPIH and either hPrp4 or hPrp18 do not dis- cyclophilin is elevated [32]. Nestel et al. [32] postulated that rupt the active site for CsA binding [29]. In addition, the NK-TR1 performs the role(s) of PPIG in NK cells due to crystal structure of PPIH [41] and PPIH in complex with a the high sequence similarity among both proteins and the ab- peptide of hPrp4 [42] reveals that the active site of PPIH re- sence of PPIG in NK cells. mains free and maintains PPIase activity when bound to hPrp4 Bourquin et al. [26] showed that the SR domain of PPIG is in vitro [42]. Taken together, these data indicate that the PPI- required for its interaction with the carboxyl terminal domain ase domain of PPIH is available to aid in folding and/or trans- (CTD) of RNA polymerase II. As a PPIase, PPIG may con- port of other spliceosomal proteins. Horowitz et al. [29] also tribute to the creation of a catalytically active form of RNA found that PPIH is part of independent complexes with polymerase II by folding the proline rich CTD [26] (Fig. 2). hPrp3/hPrp4 and hPrp18, respectively and is involved in both Although the details of the transition between transcription trans-esterification steps of splicing as part of each unique and splicing remain unknown, the participation of PPIG in assembly. In addition, CsA inhibits both trans-esterification both pathways represents an example of the intimate connec- steps of pre-mRNA splicing in vitro and in vivo, an effect that tion between these two cellular functions. The coupling of Horowitz and colleagues attribute to the association of PPIH transcription and pre-mRNA splicing has been well docu- with hPrp4 and hPrp18, respectively [29]. It is possible that mented [45]. A growing body of evidence indicates that tran- inhibition of splicing by CsA is via disruption of the PPIH scription and pre-mRNA splicing at the speckles on the PPIase active site. Both hPrp4 and hPrp18 contain a highly inner side of the nuclear membrane are structurally and func- conserved region of 31 amino acids, which is the common tionally linked, possibly for efficiency in facilitating transporta- binding site for PPIH [42]. Horowitz et al. [29] propose that tion to the cytoplasm. Proteins like PPIG may represent a PPIH mediates the proper folding and rearrangement of splice- mediator of these processes. osomal components in the early steps of splicing through an association with hPrp4 and indirectly, hPrp3 and may be in- 2.3. PPIE contains an RNA binding domain and is a part of the volved in chaperone activity or disassembly of the spliceosome human spliceosome as a member of the hPrp18 complex. The fact that PPIH forms Mi et al. [27] initially detected PPIE (also known as Cyp33 independent complexes with two splicing factors may imply and CypE in humans) in Jurkat human T-cell extracts utilizing that multiple PPIase substrates are present in the human splic- CsA affinity columns as a cyclophilin with PPIase activity and ing machinery at different times. It is well established that pep- an RNA binding domain. Subsequent sequencing of cDNAs tidyl-prolyl cis–trans isomerization is a rate-limiting step in with consensus cyclophilin domains led to the identification protein folding, and it is feasible that the introduction of a of the full-length PPIE gene [27]. This cyclophilin contains cyclophilin into the splicing machinery may contribute to the an N-terminal RNA binding domain that is able to bind efficiency of the splicing reaction. It is noteworthy that the pro- poly(A) and poly(U) tracts of nucleic acid [27]. Western blots tein with the highest amino acid identity to PPIH in Saccharo- also revealed that PPIE is exclusively present in nuclear frac- myces cerevisiae lacks the two regions diagnostic for the tions [27]. Mi et al. [27] suggest that this protein may bind spe- protein at the N-terminus of the protein and the C-terminus cific RNAs and aid in folding of additional RNA binding of helix 1 [42]. Interestingly, however, the presence of these re- proteins. More recently, two independent analyses of the hu- gions in the corresponding Cyps from a number of other spe- man spliceosome using different mass spectrometry techniques cies including Homo sapiens, Mus musculus, Drosophila identified PPIE as a member of the splicing machinery [16]. melanogaster, Caenorhabditis elegans and Arabidopsis thaliana Consistent with the functions of a number of other immuno- as well as Schizosaccharomyces pombe and other fungi suggests philins [32,29], PPIE may interact with the spliceosome to as- that these areas may have been deleted from the S. cerevisiae sist in the proper folding and/or transport of splicing factors; orthologue. If these interactions are missing in S. cerevisiae, however, further work is needed to delineate the exact role(s) the question remains whether PPIH is auxiliary or essential that this protein plays in pre-mRNA splicing. to the splicing reaction, or whether one or more PPIases substitutes for PPIH. The presence of multiple spliceosomal 2.4. PPIL1 may catalyze folding of the 35S snRNP complex immunophilins argues for a possible functional redundancy PPIL1 is a 20 kDa cyclophilin with ubiquitous expression in in the splicing system. adult human tissues [46,47]. Recent studies using mass spec- trometry have shown that PPIL1 is a component of the 45S snRNP (B1) assemblage in active splicesosomes as well as an 2.2. PPIG is a member of the transcription and splicing element of the 35S U5 snRNP [48]. The 45S aggregate contains machinery all five UsnRNAs (U1, U2, U4, U5 and U6) and more than 60 Nestel et al. [32] demonstrated that PPIG (also known as pre-mRNA splicing factors [49]. The 35S U5 snRNP contains SR-Cyp, CARS-Cyp and CYPG in humans) interacts with a large number of proteins whose association facilitates the Clk/Sty in yeast two-hybrid screens. Clk/Sty is a member of formation of the B2 spliceosomal complex (Fig. 1) [50]. PPIL1 the SR protein kinase family involved in the regulation of also binds the highly conserved transcription cofactor Ski A. Mesa et al. / FEBS Letters 582 (2008) 2345–2351 2349 interacting protein (SKIP) (Fig. 2) [47,51]. SKIP associates introns from pre-mRNA. First, as prolyl isomerases, they seem with the Sloan-Kettering virus oncoprotein (SKI), an essential to aid in folding and activation of proline containing splicing member of histone deacetylase complexes (HDAC) and repres- factors, and may be essential in assembly/disassembly of splice- sor of transforming growth factor b (TGFb) signaling [52]. osomal complexes, as well as in coupling transcription and During splicing, SKIP is recruited before the first catalytic splicing. It remains to be determined whether the immunophi- stage of splicing and remains bound through both trans-ester- lins are essential for splicing reactions to take place, if they ification reactions [47]. PPIL1 is absent during B1 assembly, possess a more auxiliary role in the splicing of particular tran- where the U4/U6.U5 tri-snRNP first comes together [50], but scripts in different tissues and/or developmental stages or both. it is incorporated during the formation of B2 (Fig. 2). Maka- The identification of immunophilins in such a ubiquitous rov et al. [48] demonstrated that PPIL1 and SKIP also associ- and universal assembly as the spliceosome may prompt us to ate with the spliced intron as part of the 35S U5 snRNP. SKIP, re-evaluate the mechanisms of action of CsA, FK506 and an indispensable spliceosomal component and transcriptional other immunosuppressive drugs that interact with immunophi- coregulator [53], may regulate the efficiency of splicing by serv- lins. For example, it is possible that some of the side effects ing as a modulator of PPIL1 [3,51]. resulting from treatments with CsA, FK506 or rapamycin These data suggest that PPIL1 plays a role during the activa- are brought about by sequestration and removal of immuno- tion of the B2 spliceosomal complex as well as the removal of philins from the spliceosome. In fact, Horowitz et al. [29] the spliced intron (Fig. 2). During the transition from the inac- found that pre-mRNA splicing is inhibited by CsA in vitro tive B1 to an active B2 spliceosome that is poised to undertake and in vivo. The association of drugs to immunophilins may the first trans-esterification reaction, PPIL1 may be recruited by displace these proteins from the splicing complex and prevent SKIP in an interaction mediated by a region other than the PPI- the creation of properly folded and active spliceosomes. Dis- ase domain of PPIL1. This would enable the PPIase active site placement of immunophilins from the spliceosome may result of PPIL1 to remain open and facilitate folding of proline rich in fewer functional splicing complexes and a subsequent reduc- members of the 45S snRNP [54]. PPIL1 may contribute to tion in mature mRNAs necessary, for example, in cell cycle the assembly and/or disassembly of the 45S snRNPs complex control or immune system activation. in a manner similar to the other spliceosomal immunophilins Understanding the exact function of the immunophilins in as a chaperone and/or folding enzyme. The presence of PPIL1 splicing may lead to the creation of novel drugs that act by as part of the 35S complex also suggests that it may associate sequestering an individual immunophilin from the spliceosome with U5 snRNP and assist the release and subsequent disman- at a particular time and/or tissue. It is clear that the highly con- tling of the 35S snRNP from the post-spliceosomal intron served nature of the PPIase domain hinders the development (Fig. 2). The discovery that two cyclophilins (PPIG and PPIL1) of ligands that will bind a single immunophilin. Therefore, it interact with both the splicing and transcription machinery al- is important to target other domains of the immunophilins, ludes to a potentially important role for these proteins in cou- as well as unique loop regions between protein motifs. In this pling the processes of transcription and splicing. These PPIases way, therapeutic agents may be directed against single pro- may bridge the two cellular functions by facilitating conforma- teins, rather than all members of a protein family. In addition, tional changes and/or transporting proteins from the machin- it is possible that some of the antigenicity against spliceosomal ery of transcription to that of the spliceosome. components in patients with autoimmune diseases resides in the cyclophilins and further examination of the epitopes 2.5. PPIL2 and PPIL3b are components of the human responsible for these diseases is necessary. Unfortunately, spliceosome work describing spliceosomal immunophilins spans a period PPIL2 (also known as Cyp60 in humans and CYP60 in of time between the mid-to late-1980s and continues only until yeast) is a 60 kDa human cyclophilin that is localized in both about 1998, after which the focus seems to have shifted to the nucleus and cytoplasm [55]. Although its specific role has other areas. Given the potential of these specific immunophi- yet to be elucidated, PPIL2 was identified as part of the human lins as possible drug receptors, further investigation is war- spliceosome using liquid chromatography tandem mass spec- ranted, specifically in relation to targetting immunophilins in trometry [16] and may serve as a chaperone or protein folding therapeutic strategies. Constant advances in molecular model- enzyme much the same way as PPIH, PPIG, PPIE and PPIL1 ing, proteomics and synthetic chemistry are sure to help pave (Fig. 2). Zhou et al. [56] identified cDNAs corresponding to the way for the development of unique and specific ligands. PPIL3b (also known as CypJ in humans) from a human fetal brain cDNA library. The PPIL3b gene encodes an alterna- References tively spliced pre-mRNA that produces two isoforms, PPIL3a [1] Moore, M.J. and Sharp, P.A. (1993) Evidence for two active sites and PPIL3b [56]. Thus far, only PPIL3b has been detected in in the splicesome provided by stereochemistry of pre-mRNA. human spliceosomes [52]. Although its exact function is un- Nature 23, 364–368. known, PPIL3b is present in the B2 complex and absent in [2] Raker, V.A., Hartmuth, K., Kastner, B. and Lu¨hrmann, R. B1, the U5 snRNP and the U4/U6.U5 tri-snRNP [31,48,50] (1999) Spliceosomal U snRNP core assembly: Sm proteins assemble onto an Sm site RNA nonanucleotide in a specific and (Fig. 2). The lack of PPIL3b in the tri-snRNP and presence thermodynamically stable manner. Mol. Cell Biol. 19, 6554–6565. in B2 suggests that it is part of the U2 snRNP complex. [3] Jurica, M.S., Licklider, L.J., Gygi, S.R., Grigorieff, N. and Moore, M.J. (2002) Purification and characterization of native spliceosomes suitable for three-dimensional structural analysis. 3. Conclusion RNA 8, 426–439. [4] Will, C.L. and Lu¨hrmann, R. (1997) Protein function in pre- mRNA splicing. Curr. Opin. Cell Biol. 9, 320–328. 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