Human MFAP1 is a cryptic ortholog of the Saccharomyces cerevisiae Spp381 splicing factor
© The Author(s). 2017
Received: 11 September 2016
Accepted: 23 February 2017
Published: 24 March 2017
Pre-mRNA splicing involves the stepwise assembly of a pre-catalytic spliceosome, followed by its catalytic activation, splicing catalysis and disassembly. Formation of the pre-catalytic spliceosomal B complex involves the incorporation of the U4/U6.U5 tri-snRNP and of a group of non-snRNP B-specific proteins. While in Saccharomyces cerevisiae the Prp38 and Snu23 proteins are recruited as components of the tri-snRNP, metazoan orthologs of Prp38 and Snu23 associate independently of the tri-snRNP as members of the B-specific proteins. The human spliceosome contains about 80 proteins that lack obvious orthologs in yeast, including most of the B-specific proteins apart from Prp38 and Snu23. Conversely, the tri-snRNP protein Spp381 is one of only five S. cerevisiae splicing factors without a known human ortholog.
Using InParanoid, a state-of-the-art method for ortholog inference between pairs of species, and systematic BLAST searches we identified the human B-specific protein MFAP1 as a putative ortholog of the S. cerevisiae tri-snRNP protein Spp381. Bioinformatics revealed that MFAP1 and Spp381 share characteristic structural features, including intrinsic disorder, an elongated shape, solvent exposure of most residues and a trend to adopt α-helical structures. In vitro binding studies showed that human MFAP1 and yeast Spp381 bind their respective Prp38 proteins via equivalent interfaces and that they cross-interact with the Prp38 proteins of the respective other species. Furthermore, MFAP1 and Spp381 both form higher-order complexes that additionally include Snu23, suggesting that they are parts of equivalent spliceosomal sub-complexes. Finally, similar to yeast Spp381, human MFAP1 partially rescued a growth defect of the temperature-sensitive mutant yeast strain prp38-1.
Human B-specific protein MFAP1 structurally and functionally resembles the yeast tri-snRNP-specific protein Spp381 and thus qualifies as its so far missing ortholog. Our study indicates that the yeast Snu23-Prp38-Spp381 triple complex was evolutionarily reprogrammed from a tri-snRNP-specific module in yeast to the B-specific Snu23-Prp38-MFAP1 module in metazoa, affording higher flexibility in spliceosome assembly and thus, presumably, in splicing regulation.
KeywordsAlternative splicing B-specific proteins Pre-mRNA splicing Spliceosome U4/U6.U5 Tri-snRNP-specific proteins
Splicing of primary transcripts is an essential step in the expression of many eukaryotic protein-coding genes. During splicing, non-coding intervening sequences (introns) are excised from a precursor (pre-) mRNA and neighboring coding regions (exons) are ligated via two consecutive transesterification reactions [1, 2]. Pre-mRNA splicing is catalyzed by the spliceosome, a highly dynamic, multi-megadalton molecular ribonucleoprotein (RNP) machine that is composed of five small nuclear (sn) RNPs and numerous non-snRNP proteins [3, 4]. For each round of splicing, a spliceosome is assembled in a stepwise fashion. The vast majority of splicing events in Saccharomyces cerevisiae (sc) is constitutive and involves assembly of a spliceosome across an intron. In a constitutive splice event, U1 and U2 snRNPs recognize the 5’-splice site and branch point sequence of an intron, respectively, forming the A complex. Subsequently, the U4, U5 and U6 snRNPs join as a pre-formed tri-snRNP, giving rise to the pre-catalytic B complex. The B complex is then catalytically activated, yielding first the Bact and subsequently the B* complex. The latter can carry out the first transesterification reaction of a splicing event. After step one of splicing, further rearrangements give rise to the C complex, which catalyzes the second transesterification reaction, subsequent to which the spliceosome is disassembled and subunits are recycled [3, 4].
Most primary transcripts in complex, multicellular eukaryotes contain more than one intron and can undergo alternative splicing to yield multiple mature mRNAs originating from the same gene . The lengths of their introns vary considerably and can amount to several hundreds of thousands of nucleotides , while their exons are on average much shorter (ca. 120 nucleotides) and more homogeneous in size [7, 8]. Therefore, faithful localization of authentic 5’- and 3’-splice sites in complex, multicellular organisms is thought to occur via the initial assembly of spliceosomal complexes across exons (exon definition), which commits the pre-mRNA to the splicing pathway [9–12]. To allow intron excision, the interactions established during exon definition have to be reorganized to allow a 3’-splice site to be paired with an upstream 5’-splice site. Exon definition may proceed either to a cross-intron A complex  or directly to a cross-intron B complex under omission of a cross-intron A stage . Functional pairing of specific splice sites, and thus the decision on a certain splicing pattern, is thought to take place during this conversion of cross-exon to cross-intron spliceosomal complexes [10, 14–16].
In yeast, pre-mRNA processing factor 38 domain containing protein (Prp38) and 23 kDa small nuclear ribonucleoprotein component (Snu23) are integral components of the U4/U6.U5 tri-snRNP [17, 18], stay associated during tri-snRNP integration and B complex formation and leave the spliceosome again during the transition to the Bact complex . The human orthologs of Prp38 and Snu23 are also exclusively present at the B complex stage but, in contrast to their yeast orthologs, associate with the pre-catalytic spliceosome independent of the tri-snRNP . This feature they share with seven other non-snRNP proteins, collectively referred to as B-specific proteins. The specific recruitment of B-specific proteins to the spliceosome during cross-exon to cross-intron switching makes them prime candidates as regulators of alternative splicing.
Indeed, for most B-specific proteins there is evidence that they play a role in alternative splicing. In human, the group of B-specific proteins includes Prp38, Snu23, microfibrillar-associated protein 1 (MFAP1), suppressor of mec-8 and unc-52 protein homolog 1 (Smu1), Arg-Glu/Asp-repeat-containing protein (RED), formin-binding protein 21 (FBP21), 38 kDa nuclear protein containing a WW domain (NPW38), NPW38-binding protein (NPW38BP) and ubiquitin-like protein 5 (UBL5) . Homo sapiens (hs) Prp38 has acquired a veritable, C-terminal arginine-serine-rich (RS) domain, a hallmark of the splicing regulatory serine-arginine-rich (SR) proteins that are largely lacking in yeast . UBL5, also called Hub1, was the first splicing factor that was found to be involved in alterative splicing in human  as well as in a rare case of alternative splicing in yeast . In contrast, MFAP1, Smu1, RED, FBP21, NPW38 and NPW38BP lack obvious orthologs in yeast, where alternative splicing is essentially absent. MFAP1, Smu1 and RED have been implicated directly in the modulation of splice site choices in certain pre-mRNAs [24–29].
Presently, the precise functions of B-specific proteins are unknown. In particular, it is not clear to which extent they are important for both constitutive and alternative splicing, whether orthologs of some of these proteins are truly missing in yeast or have evolved so that they are not easily recognized or if yeast harbors other splicing factors that take over the constitutive roles of some of the B-specific proteins.
MFAP1 was first identified as a component of the extracellular matrix . Later, the protein was found in spliceosome preparations , was shown to interact with Prp38 in pull-down experiments and to be required for pre-mRNA processing . Interactions between MFAP1 and other B-specific proteins were identified by yeast two-hybrid (Y2H) [21, 33] and in vitro binding studies [21, 34]. Due to its elongated, solvent exposed nature and predicted dense array of short protein binding motifs, MFAP1 was suggested to act as a scaffold or ruler that engages multiple binding partners . Recently, the molecular details of the MFAP1-Prp38 interaction have been revealed by X-ray crystallography , representing one of the few structurally characterized interactions between B-specific proteins besides Snu23-Prp38 and Smu1-RED .
Here, we investigated whether S. cerevisiae contains an ortholog of metazoan MFAP1. Using InParanoid 8 and systematic BLAST searches, multiple sequence alignments, structure-guided interaction studies and a yeast growth assay, we identified the tri-snRNP-specific pre-mRNA-splicing factor, suppressor of prp38-1 (Spp381), as the so far missing MFAP1 ortholog in S. cerevisiae.
Identification of MFAP1 orthologs in the eukaryotic tree of life
To investigate when in eukaryotic evolution an MFAP1-coding gene has been acquired, we conducted an ortholog search using the InParanoid 8 orthology analysis tool [36, 37]. The InParanoid methodology  uses pairwise BLAST-based all-versus-all sequence comparisons to detect orthologs in sets of protein-coding genes from 273 species (covering the major branches of the eukaryotic tree of life and selected prokaryotes), with each gene represented by one protein. To exclude false positive hits that merely arise from co-occurrence of abundant, highly conserved domains, InParanoid uses a strict cut-off criterion of sequence coverage ≥ 50% and BLAST score ≥ 50. Taking into account the presumably low sequence conservation of MFAP1 due to the predicted structural disorder and the absence of folded protein domains , we also performed reciprocal best BLAST hit (RBH) searches of MFAP1 proteins against the same 273 sets of protein-coding genes with relaxed cut-off criteria (BLAST score ≥ 30, E-value ≤ 0.01). The RBH method has a relatively high specificity compared to other ortholog detection methods and its specificity is only marginally affected by changes in cut-off values .
Stepwise BLAST searches focused on the fungal kingdom identify Spp381 as a potential MFAP1 ortholog in Saccharomycetaceae
As expected, MFAP1 orthologs were detected in the majority of non-Saccharomycotina fungi (81.0%, 64/79), as well as in several non-Saccharomycetaceae Saccharomycotina species (54.5%, 6/11), but were specifically absent in Saccharomycetaceae (0%, 0/14). We assumed that if MFAP1 orthologs exist in Saccharomycetaceae, they would be evolutionary closest to neighboring Saccharomycotina species. Thus, we repeated the BLAST search with hsMFAP1 orthologs identified in the Saccharomycotina species Yarrowia lipolytica (yl, UniProt ID: Q6CA21), Pichia pastoris (pp, UniProt ID: A0A1B2J9D1), Debaryomyces hansenii (dh, UniProt ID: Q6BII8) and Candida albicans (ca, UniProt ID: C4YG44) against the 25 Saccharomycotina species of the Medina et al. fungal tree of life. All four species identified MFAP1 orthologs in the majority of non-Saccharomycetaceae Saccharomycotina species (yl: 7/11; pp: 10/11; dh: 9/11; ca: 11/11). In addition, all four also identified an MFAP1 ortholog in the Saccharomycetaceae organism Kluyveromyces lactis (Spp381, UniProt ID: Q6CJ60). Furthermore, Saccharomycetaceae MFAP1 orthologs were identified in Candida glabrata (UniProt ID: Q6FU95) by dhMFAP1 and in Lachancea thermotolerans by ppMFAP1, besides six medium confidence hits (query coverage 10–20%) by ppMFAP1. We next selected K. lactis and C. glabrata MFAP1 orthologs as queries. Both queries identified orthologs in the same nine of 14 Saccharomycetaceae species, including Saccharomyces cerevisiae (Spp381, UniProt ID: P38282). In addition, the C. glabrata protein identified an ortholog in P. pastoris and K. lactis Spp381 found orthologs in most non-Saccharomycetaceae Saccharomycotina species (9/11). Finally, we performed the same analysis with the identified S. cerevisiae protein Spp381 as query and found orthologs in the same Saccharomycetaceae species (9/14) as with P. pastoris and C. glabrata in addition to one hit in non-Saccharomycetaceae Saccharomycotina (in P. pastoris). While the overall low sequence conservation of MFAP1 orthologs is especially pronounced between Saccharomycetaceae and neighboring Saccharomycotina species, the sequences of the MFAP1 orthologs of K. lactis (Spp381) and P. pastoris are able to bridge this gap.
To test if Spp381 proteins found in Saccharomycetaceae indeed represent a group of MFAP1 orthologs and not a different protein that coexists in MFAP1-containing non-Saccharomycetceae species, we used Spp381 from S. cerevisiae as a query in our InParanoid-based ortholog search (Fig. 1). ScSpp381 yielded orthologs in Mycospaerella graminicola, Sclerotinia sclerotiorum (both non-Saccharomycotina Ascomycota), Pichia pastoris (non-Saccharomycetaceae Saccharomycotina) – these proteins are the same as those identified in the initial search with hsMFAP1 – and in all twelve Saccharomycetaceae species. Thus, we did not find any non-MFAP1 protein as an scSpp381 ortholog. These results show that Spp381 is not closely related, according to the InParanoid cut-off criteria, to any non-MFAP1 protein outside Saccharomycetaceae, indicating that Spp381 and MFAP1 do not coexist as different proteins in non-Saccharomycetaceae species. However, it is still possible that MFAP1 and Spp381 are highly similar proteins that emerged by convergent evolution and that exist in exactly complementary groups of organisms. It also cannot be excluded that MFAP1 and Spp381 might have emerged from the same ancestral gene by duplication (paralogs) and that a different copy was lost in Saccharomycetaceae (mfap1) versus non-Saccharomycetaceae (spp381).
S. cerevisiae Spp381 shares physicochemical, biochemical and structural features with hsMFAP1
Interaction studies corroborate similar functions of scSpp381 and hsMFAP1
Human MFAP1 can partially substitute for yeast Spp381 in its function to rescue the conditionally lethal mutant yeast strain prp38-1
HsMFAP1 is a cryptic ortholog of the yeast splicing factor Spp381
Proteomics analyses revealed that almost all factors required for constitutive splicing in S. cerevisiae are also present in human spliceosomes [4, 19]. Presently, yeast proteins with missing human orthologs include the U1 factors Prp42 and Snu56, the Prp19-associated complex protein Ntc20, the disassembly factor Ntr2  and the U4/U6.U5 tri-snRNP-specific protein Spp381. Compared to yeast, human spliceosomes include ~ 80 additional, predominantly non-snRNP proteins, whose precise functions during splicing are in many cases unclear [4, 19].
Summary of ortholog analyses
Taken together, the sequence similarity between MFAP1 and SPP381 does not suffice to delineate their precise evolutionary relationships. Yet, they are structurally and functionally similar to an extent that they can substitute for each other. This suggests that, indeed, both proteins may represent orthologs although other evolutionary scenarios cannot be entirely ruled out.
Functional characteristics of MFAP1 and Spp381 proteins may allow for high evolutionary rates of sequence divergence
Identification of a common evolutionary origin of proteins by sequence comparisons is increasingly challenging with decreasing sequence conservation. Fast diverging sequences lack the evolutionary pressure commonly associated with the maintenance of a particular 3D fold or of extended interaction surfaces. The human B-specific protein MFAP1 is characterized by a lack of stable tertiary structure, structural flexibility and relatively short, but nevertheless high-affine, protein-protein interaction sites and plays a role as an elongated scaffolding factor that could transmit conformational changes within the spliceosome . These functional characteristics likely allow for a high sequence divergence rate during evolution, in particular in regions of the protein that only require the maintenance of an elongated, flexible structure.
Indeed, the sequence identity between known MFAP1 orthologs is low and even less recognizable for evolutionary distant MFAP1 orthologs identified in our study (Additional file 5). In this context it is not surprising that MFAP1 and Saccharomycetaceae Spp381 sequences also exhibit a low sequence identity. More surprising, however, is the low sequence conservation between Saccharomycotina and other Ascomycota species, between Saccharomycotina and Saccharomycetaceae, and even between neighboring Saccharomycetaceae organisms (Additional file 5).
Liberation from the tri-snRNP may enable B-specific proteins to perform their functions in a regulated manner
Our findings suggest that a similar functional relationship as between yeast and metazoan Prp38 and Snu23 proteins  exists between yeast Spp381 and metazoan MFAP1 proteins. As disruption of the scspp381 gene leads to severe growth defects and accumulation of unspliced pre-mRNA in vivo , scSpp381 is an important, albeit not essential, splicing factor that apparently acts in the same process as scPrp38. We showed that scSpp381 and hsMFAP1 exhibit cross-species interactions with the respective Prp38 proteins, suggesting that MFAP1 may be responsible for Spp381-like functions in complex, multicellular eukaryotes. During functional pairing of splice sites after initial cross-intron or cross-exon spliceosome assembly, spliceosomes face the problem of locating and bringing together spliceosomal subunits that are bound at the intron ends and thus may be spatially separated . Elongated proteins that are specifically recruited at this stage, such as hsMFAP1 and scSpp381, are well suited to help align and gather spatially separated parts of the spliceosome. They could serve as scaffolds or rulers, e.g. during functional pairing of splice sites, by using limited-length binding epitopes arrayed along their sequence to engage multiple binding partners . However, like scPrp38 and scSnu23, scSpp381 is a stable component of the U4/U6.U5 tri-snRNP [17, 18, 43], while hsMFAP1, like hsPrp38 and hsSnu23, is a non-snRNP B-specific protein  (Fig. 9). As in the case of yeast and metazoan Prp38 and Snu23, the tri-snRNP nature of scSpp381 mandates that it is always recruited to spliceosomal B complexes together with other tri-snRNP components, thus rendering its function constitutive. In contrast, the non-snRNP, B-specific MFAP1 protein could be differentially recruited in different, mutually exclusive, splicing situations. Such variable recruitment could influence the relative efficiencies with which competing, alternative splice events are carried out.
Our study revealed the so far uncharted evolutionary backgrounds of the H. sapiens B-specific protein MFAP1 and of the S. cerevisiae tri-snRNP protein Spp381. Prior to this work, MFAP1 was thought to exclusively exist in spliceosomes of complex, multicellular organisms. We have shown that an MFAP1 ortholog is present not only in S. cerevisiae but also in organisms that separated from the common lineage with complex, multicellular eukaryotes about 1.8 billion years ago. Spp381 was suggested to be one of only five yeast splicing factors without a human ortholog. Its evolutionary connection to MFAP1 reduces this number to four, raising the question if finally all ancient yeast splicing factors turn out to be conserved in complex, multicellular eukaryotes. As exemplified by the present study, identifying evolutionary connections between proteins may point to potential functions as well as potential interaction partners of poorly characterized proteins.
Automated search for orthologs by InParanoid 8
Ortholog searches were conducted using InParanoid 8 [36, 37]. InParanoid 8 is based on sets of protein-coding genes of 273 species, where each gene is represented by one protein. These species include the 66 reference species that the ‘Quest for Orthologs’ community has agreed on using plus 207 additional species with completely sequenced genomes and cover all major branches of the eukaryotic tree of life (246 species) and a representative selection of 27 prokaryotes. The InParanoid methodology  uses a pairwise BLAST-based all-versus-all sequence comparison to detect orthologs. If candidate sequences are orthologs, they should score higher with each other than with any other sequence in the other organism’s set of protein-coding genes. InParanoid further applies special cluster analysis rules to extract all in-paralogs and exclude all out-paralogs . InParanoid uses a strict cut-off criterion of sequence coverage ≥ 50% and BLAST score ≥ 50. The InParanoid 8 ortholog database [36, 37] provides a user interface to find orthologs inferred by the InParanoid algorithm.
Secondly, we performed RBH searches with different MFAP1 or Spp381 protein sequences against the same sets of protein-coding genes of the 273 species selected by InParanoid using the InParanoid web server . A BLAST hit was considered an ortholog if the BLAST score was ≥ 30 with E-value ≤ 0.01, and if the reverse BLAST search, i.e. the BLAST hit was used as query in a BLAST search against the set of protein-coding genes of the original query’s organism, resulted the initial query protein as the best hit. This search aims to identify orthologs that do not survive the strict cut-off criteria used for the InParanoid 8 database .
Manual search for orthologs focused on the fungal kingdom
For an MFAP1 ortholog search among the fungi, we performed individual BLAST searches with Homo sapiens MFAP1 (UniProt ID: P55081) as a query against the proteomes of 103 fungal species that represent the fungal tree of life as published by Medina et al. . Seven MFAP1 orthologs identified in the Saccharomycotina subphylum, i.e. MFAP1 orthologs of Yarrowia lipolytica (UniProt ID: Q6CA21), Pichia pastoris (UniProt ID: A0A1B2J9D1), Debaryomyces hansenii (UniProt ID: Q6BII8), Candida albicans (UniProt ID: C4YG44), Kluyveromyces lactis (UniProt ID: Q6CJ60), Candida glabrata (UniProt ID: Q6FU95) and Saccharomyces cerevisiae Spp381 (UniProt ID: P38282), were then used as query sequences in further individual BLAST searches against the 25 Saccharomycotina species, including 14 Saccharomycetaceae species, that are part of the 103 fungal species. A BLAST hit was considered an ortholog of the query protein if the BLAST score (calculated with the BLOSUM45 scoring matrix) was ≥ 30 with an E-value ≤ 0.01 and query coverage ≥ 20% (high confidence) or ≥ 10% (medium confidence), and if the reverse BLAST search resulted in the initial query protein as the best hit.
Generation of multiple sequence alignment of MFAP1 orthologs
Pairwise sequence alignment
Sequence identity and sequence similarity values were obtained from pairwise sequence alignments by the EMBOSS Needle tool  using a BLOSUM62 scoring matrix.
Protein sequence analyses
The PredictProtein package  was used for secondary structure (REPROFSec), solvent exposure (PROFAcc) and structural disorder (Meta-Disorder) predictions.
Plasmids for recombinant protein production in E. coli
Open reading frames (ORFs) encoding hsPrp38 or hsMFAP1 were amplified from a human cDNA library and cloned into the pETM11 vector using EMP cloning as described . ORFs encoding scPrp38 and scSpp381 were PCR-amplified from S. cerevisiae genomic DNA and cloned into the pETM11 vector using EMP cloning . Truncations and point mutations were introduced by inverse PCR as described . The pETM11 vector guides the production of amino-terminally His6-tagged, TEV-cleavable fusion proteins.
Protein production and purification
Proteins bearing an N-terminal, TEV-cleavable His6-tag were produced in E. coli Rosetta 2 (DE3) or E. coli BL21 (DE3) RIL cells in auto-inducing ZY medium  for 24 h at 18 °C. The following steps were performed at 4 °C. Cells were resuspended in solubilization buffer (50 mM sodium phosphate, pH 8.0, 500 mM NaCl, 30 mM imidazole, 5 mM β-mercaptoethanol) and lyzed using an EmulsiFlex-C5 cell homogenizer (Avestin). The soluble fraction was separated from the insoluble fraction by centrifugation for 30 min at 55,900 x g in an Avanti J-26 XP centrifuge (Beckman Coulter). Target proteins were captured on Ni2+-NTA resin (GE Healthcare), washed with solubilization buffer and eluted with elution buffer (250 mM imidazole, pH 8.0, 300 mM NaCl, 5 mM β-mercaptoethanol). Tags were cleaved with 1:50 TEV during overnight dialysis against 10 mM sodium phosphate, pH 8.0, 300 mM NaCl, 30 mM imidazole, 5 mM β-mercaptoethanol, and cleaved samples were again passed over Ni2+-NTA resin. The flow-through was collected, concentrated, and subjected to size exclusion chromatography (SEC) in SEC buffer (10 mM Tris-HCl, pH 8.0, 300 mM NaCl, 0.1 mM EDTA, 1 mM DTT) using Superdex 75 and Superdex 200 columns (GE Healthcare). Peak fractions were analyzed by SDS-PAGE. Fractions containing the target protein were pooled, concentrated, and shock-frozen in liquid nitrogen.
Analytical gel filtration chromatography
Proteins (50 μM), alone or with an equimolar amount of binding partner, were incubated in SEC buffer for 30 min at 4 °C. 50 μl of sample were analyzed on Superdex 75 PC 3.2/30 or Superdex 200 Increase 3.2/300 size exclusion columns (GE Healthcare) using an ÄKTAmicro system (GE Healthcare) at 4 °C. The peak fractions were inspected by SDS-PAGE.
Circular dichroism spectroscopy
Proteins were dialyzed against CD buffer (10 mM sodium phosphate, pH 8.0, 50 mM sodium perchlorate) at 4 °C overnight, and diluted to a final concentration of 4.5 μM (hsMFAP130-344) or 5.1 μM (scSpp381FL). All spectra were recorded with a Jasco J-810 spectropolarimeter using quartz cuvettes with 0.2 mm path length. Initial CD spectra were collected at wavelengths between 190 and 240 nm at 4 °C. CD melting profiles were then recorded by heating the samples to 90 °C at a rate of 2 °C/min and following the CD signal at 222 nm. Final CD spectra were measured at wavelengths between 190 and 240 nm at 90 °C.
Yeast strains and yeast plasmids
Yeast strains used in this study are MGD353-46D (MATα leu2-3,112 trp1-289 ura3-52 his3(-) cyh r ) and ts192 (MATα prp38-1 leu2-3,112 trp1-289 ura3-52 his3(-) cyh r ), kindly provided by Brian C. Rymond (University of Kentucky). Yeast plasmids used in this study are YEp13-2 (prp38, leu2, amp r ), YEp13-7 (spp381, leu2, amp r ), YEplac112-7A (spp381, trp1, amp r ), all kindly provided by Brian C. Rymond (University of Kentucky), and YEplac112-MFAP1 (mfap1, trp1, amp r ). YEplac112-MFAP1 was produced by using YEplac112-7A as a template and replacing the scSpp381 coding region with the coding region of hsMFAP1 by EMP cloning .
For generation of electro-competent S. cerevisiae cells, a 50 ml YPD culture was inoculated with overnight culture to an OD600 of 0.1 and grown at 30 °C and 250 rpm to an OD600 of 1.5–10. Cells were harvested by centrifugation for 10 min at 2,000 × g and 4 °C, resuspended in 10 ml YPD, 2 ml 1 M HEPES, pH 8.0, 250 μl 1 M DTT, and incubated for 15 min at 30 °C and 250 rpm. Cells were resuspended in 50 ml of ice-cold milliQ H2O and again centrifuged for 10 min at 2,000 × g, 4 °C. Subsequently, cells were washed with 2 ml ice-cold 1 M sorbitol and centrifuged for 10 min at 2,000 × g and 4 °C. Finally, cells were resuspended in 500 μl ice-cold 1 M sorbitol, aliquoted, and directly used for transformation.
Two microgram plasmid were mixed with 50 μl electro-competent S. cerevisiae cells and incubated for 15 min on ice. Subsequent to the electric shock at 1,500 V, 500 μl of ice-cold 1 M sorbitol were added and cells were incubated for 2 h at 30 °C and 250 rpm. For selection of plasmid-containing cells, the cell suspension was plated on minimal medium agar plates lacking leucine (in case of YEplac13 plasmids) or tryptophan (in case of YEplac112 plasmids).
Yeast growth assay
Yeast strains were grown overnight in liquid minimal medium (6.8 g/l yeast nitrogen base without amino acids, 20 g/l glucose, 40.0 mg/l adenine, 19.2 mg/l uracil, 19.2 mg/l L-arginine, 96.0 mg/l L-aspartic acid, 96.0 mg/l L-glutamic acid, 19.2 mg/l L-histidine, 28.8 mg/l L-lysine, 19.2 mg/l L-methionine, 48.0 mg/l L-phenylalanine, 360.0 mg/l L-serine, 192.0 mg/l L-threonine, 14.4 mg/l L-tyrosine, 144.0 mg/l L-valine, and for YEplac112 plasmid-containing strains 57.6 mg/l L-leucine, for YEp13 plasmid-containing strains 38.4 mg/l L-tryptophan, and for strains without plasmid 57.6 mg/l L-leucine and 38.4 mg/l L-tryptophan) at 30 °C and 250 rpm. Subsequently, cultures were diluted to an OD600 of 2.0, 0.2, 0.02, and 0.002. 5 μl of each dilution were spotted on YPD-agar plates and plates were incubated at 23 or 37 °C for 3 days.
- ca :
- ce :
- ct :
- dh :
- hs :
- kl :
Microfibrillar-associated protein 1
- pp :
pre-mRNA processing factor 38 domain containing protein
Reciprocal best BLAST hit
- RS domain:
- sc :
Sodium dodecyl sulfate polyacrylamide gel electrophoresis
Size exclusion chromatography
small nuclear ribonucleoprotein
23 kDa small nuclear ribonucleoprotein component
pre-mRNA-splicing factor suppressor of prp38-1
- SR protein:
- wc :
- yl :
We thank Thomas Stellwag (Freie Universität Berlin) and Patrick Knox (Beuth Hochschule für Technik) for help with cloning and purification of truncated scSpp381 and scSnu23 variants, Junqiao Jia and Ronja Janke (both Freie Universität Berlin) for advice with the yeast growth assay and Brian C. Rymond (University of Kentucky) for kindly providing yeast strains and yeast plasmids.
This work was supported by the Deutsche Forschungsgemeinschaft (grant WA 1126/7-1 to MCW).
Availability of data and materials
All data generated or analyzed during this study are included in this published article and its supplementary information file.
Conceived and designed the experiments: AKCU, MCW. Performed the experiments: AKCU. Analyzed and interpreted the data: AKCU, MCW. Wrote the manuscript: AKCU, MCW. Approved the manuscript: AKCU, MCW.
The authors declare that they have no competing interests.
Consent for publication
Ethics approval and consent to participate
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- Moore MJ, Sharp PA. Evidence for two active sites in the spliceosome provided by stereochemistry of pre-mRNA splicing. Nature. 1993;365(6444):364–8.View ArticlePubMedGoogle Scholar
- Padgett RA, Grabowski PJ, Konarska MM, Seiler S, Sharp PA. Splicing of messenger RNA precursors. Annu Rev Biochem. 1986;55:1119–50.View ArticlePubMedGoogle Scholar
- Wahl MC, Will CL, Luhrmann R. The spliceosome: design principles of a dynamic RNP machine. Cell. 2009;136(4):701–18.View ArticlePubMedGoogle Scholar
- Will CL, Luhrmann R. Spliceosome structure and function. Cold Spring Harb Perspect Biol. 2011;3(7).
- Graveley BR. Alternative splicing: increasing diversity in the proteomic world. Trends Genet. 2001;17(2):100–7.View ArticlePubMedGoogle Scholar
- Deutsch M, Long M. Intron-exon structures of eukaryotic model organisms. Nucleic Acids Res. 1999;27(15):3219–28.View ArticlePubMedPubMed CentralGoogle Scholar
- Ast G. How did alternative splicing evolve? Nat Rev Genet. 2004;5(10):773–82.View ArticlePubMedGoogle Scholar
- Hawkins JD. A survey on intron and exon lengths. Nucleic Acids Res. 1988;16(21):9893–908.View ArticlePubMedPubMed CentralGoogle Scholar
- Berget SM. Exon recognition in vertebrate splicing. J Biol Chem. 1995;270(6):2411–4.View ArticlePubMedGoogle Scholar
- Lim SR, Hertel KJ. Commitment to splice site pairing coincides with a complex formation. Mol Cell. 2004;15(3):477–83.View ArticlePubMedGoogle Scholar
- Michaud S, Reed R. An ATP-independent complex commits pre-mRNA to the mammalian spliceosome assembly pathway. Genes Dev. 1991;5(12B):2534–46.View ArticlePubMedGoogle Scholar
- Reed R. Mechanisms of fidelity in pre-mRNA splicing. Curr Opin Cell Biol. 2000;12(3):340–5.View ArticlePubMedGoogle Scholar
- Schneider M, Will CL, Anokhina M, Tazi J, Urlaub H, Luhrmann R. Exon definition complexes contain the tri-snRNP and can be directly converted into B-like precatalytic splicing complexes. Mol Cell. 2010;38(2):223–35.View ArticlePubMedGoogle Scholar
- Bonnal S, Martinez C, Forch P, Bachi A, Wilm M, Valcarcel J. RBM5/Luca-15/H37 regulates Fas alternative splice site pairing after exon definition. Mol Cell. 2008;32(1):81–95.View ArticlePubMedGoogle Scholar
- House AE, Lynch KW. An exonic splicing silencer represses spliceosome assembly after ATP-dependent exon recognition. Nat Struct Mol Biol. 2006;13(10):937–44.View ArticlePubMedGoogle Scholar
- Sharma S, Kohlstaedt LA, Damianov A, Rio DC, Black DL. Polypyrimidine tract binding protein controls the transition from exon definition to an intron defined spliceosome. Nat Struct Mol Biol. 2008;15(2):183–91.View ArticlePubMedPubMed CentralGoogle Scholar
- Gottschalk A, Neubauer G, Banroques J, Mann M, Luhrmann R, Fabrizio P. Identification by mass spectrometry and functional analysis of novel proteins of the yeast [U4/U6.U5] tri-snRNP. EMBO J. 1999;18(16):4535–48.View ArticlePubMedPubMed CentralGoogle Scholar
- Stevens SW, Abelson J. Purification of the yeast U4/U6.U5 small nuclear ribonucleoprotein particle and identification of its proteins. Proc Natl Acad Sci U S A. 1999;96(13):7226–31.View ArticlePubMedPubMed CentralGoogle Scholar
- Fabrizio P, Dannenberg J, Dube P, Kastner B, Stark H, Urlaub H, Luhrmann R. The evolutionarily conserved core design of the catalytic activation step of the yeast spliceosome. Mol Cell. 2009;36(4):593–608.View ArticlePubMedGoogle Scholar
- Agafonov DE, Deckert J, Wolf E, Odenwalder P, Bessonov S, Will CL, Urlaub H, Luhrmann R. Semiquantitative proteomic analysis of the human spliceosome via a novel two-dimensional gel electrophoresis method. Mol Cell Biol. 2011;31(13):2667–82.View ArticlePubMedPubMed CentralGoogle Scholar
- Schutze T, Ulrich AK, Apelt L, Will CL, Bartlick N, Seeger M, Weber G, Luhrmann R, Stelzl U, Wahl MC. Multiple protein-protein interactions converging on the Prp38 protein during activation of the human spliceosome. RNA. 2016;22(2):265–77.View ArticlePubMedPubMed CentralGoogle Scholar
- Ammon T, Mishra SK, Kowalska K, Popowicz GM, Holak TA, Jentsch S. The conserved ubiquitin-like protein Hub1 plays a critical role in splicing in human cells. J Mol Cell Biol. 2014;6(4):312–23.View ArticlePubMedPubMed CentralGoogle Scholar
- Mishra SK, Ammon T, Popowicz GM, Krajewski M, Nagel RJ, Ares Jr M, Holak TA, Jentsch S. Role of the ubiquitin-like protein Hub1 in splice-site usage and alternative splicing. Nature. 2011;474(7350):173–8.View ArticlePubMedPubMed CentralGoogle Scholar
- Chung T, Wang D, Kim CS, Yadegari R, Larkins BA. Plant SMU-1 and SMU-2 homologues regulate pre-mRNA splicing and multiple aspects of development. Plant Physiol. 2009;151(3):1498–512.View ArticlePubMedPubMed CentralGoogle Scholar
- Ma L, Gao X, Luo J, Huang L, Teng Y, Horvitz HR. The Caenorhabditis elegans gene mfap-1 encodes a nuclear protein that affects alternative splicing. PLoS Genet. 2012;8(7):e1002827.View ArticlePubMedPubMed CentralGoogle Scholar
- Spartz AK, Herman RK, Shaw JE. SMU-2 and SMU-1, Caenorhabditis elegans homologs of mammalian spliceosome-associated proteins RED and fSAP57, work together to affect splice site choice. Mol Cell Biol. 2004;24(15):6811–23.View ArticlePubMedPubMed CentralGoogle Scholar
- Spike CA, Shaw JE, Herman RK. Analysis of smu-1, a gene that regulates the alternative splicing of unc-52 pre-mRNA in Caenorhabditis elegans. Mol Cell Biol. 2001;21(15):4985–95.View ArticlePubMedPubMed CentralGoogle Scholar
- Sugaya K, Hongo E, Ishihara Y, Tsuji H. The conserved role of Smu1 in splicing is characterized in its mammalian temperature-sensitive mutant. J Cell Sci. 2006;119(Pt 23):4944–51.View ArticlePubMedGoogle Scholar
- Sugaya K, Ishihara Y, Sugaya K. Enlargement of speckles of SF2/ASF due to loss of function of Smu1 is characterized in the mammalian temperature-sensitive mutant. RNA Biol. 2011;8(3):488–95.View ArticlePubMedGoogle Scholar
- Horrigan SK, Rich CB, Streeten BW, Li ZY, Foster JA. Characterization of an associated microfibril protein through recombinant DNA techniques. J Biol Chem. 1992;267(14):10087–95.PubMedGoogle Scholar
- Neubauer G, King A, Rappsilber J, Calvio C, Watson M, Ajuh P, Sleeman J, Lamond A, Mann M. Mass spectrometry and EST-database searching allows characterization of the multi-protein spliceosome complex. Nat Genet. 1998;20(1):46–50.View ArticlePubMedGoogle Scholar
- Andersen DS, Tapon N. Drosophila MFAP1 is required for pre-mRNA processing and G2/M progression. J Biol Chem. 2008;283(45):31256–67.View ArticlePubMedPubMed CentralGoogle Scholar
- Hegele A, Kamburov A, Grossmann A, Sourlis C, Wowro S, Weimann M, Will CL, Pena V, Luhrmann R, Stelzl U. Dynamic protein-protein interaction wiring of the human spliceosome. Mol Cell. 2012;45(4):567–80.View ArticlePubMedGoogle Scholar
- Ulrich AK, Seeger M, Schutze T, Bartlick N, Wahl MC. Scaffolding in the spliceosome via single alpha helices. Structure. 2016;24(11):1972–83.View ArticlePubMedGoogle Scholar
- Ulrich AK, Schulz JF, Kamprad A, Schutze T, Wahl MC. Structural basis for the functional coupling of the alternative splicing factors Smu1 and RED. Structure. 2016;24(5):762–73.View ArticlePubMedGoogle Scholar
- InParanoid8. http://InParanoid.sbc.su.se. Accessed 01 Nov 2016.
- Sonnhammer EL, Ostlund G. InParanoid 8: orthology analysis between 273 proteomes, mostly eukaryotic. Nucleic Acids Res. 2015;43(Database issue):D234–9.View ArticlePubMedGoogle Scholar
- Remm M, Storm CE, Sonnhammer EL. Automatic clustering of orthologs and in-paralogs from pairwise species comparisons. J Mol Biol. 2001;314(5):1041–52.View ArticlePubMedGoogle Scholar
- Chen F, Mackey AJ, Vermunt JK, Roos DS. Assessing performance of orthology detection strategies applied to eukaryotic genomes. Plos One. 2007;2(4):e383.View ArticlePubMedPubMed CentralGoogle Scholar
- TimeTree.org. http://www.timetree.org. Accessed 25 Jan 2017.
- Hedges SB, Dudley J, Kumar S. TimeTree: a public knowledge-base of divergence times among organisms. Bioinformatics. 2006;22(23):2971–2.View ArticlePubMedGoogle Scholar
- Medina EM, Jones GW, Fitzpatrick DA. Reconstructing the fungal tree of life using phylogenomics and a preliminary investigation of the distribution of yeast prion-like proteins in the fungal kingdom. J Mol Evol. 2011;73(3–4):116–33.View ArticlePubMedGoogle Scholar
- Lybarger S, Beickman K, Brown V, Dembla-Rajpal N, Morey K, Seipelt R, Rymond BC. Elevated levels of a U4/U6.U5 snRNP-associated protein, Spp381p, rescue a mutant defective in spliceosome maturation. Mol Cell Biol. 1999;19(1):577–84.View ArticlePubMedPubMed CentralGoogle Scholar
- Blanton S, Srinivasan A, Rymond BC. PRP38 encodes a yeast protein required for pre-mRNA splicing and maintenance of stable U6 small nuclear RNA levels. Mol Cell Biol. 1992;12(9):3939–47.View ArticlePubMedPubMed CentralGoogle Scholar
- Xie J, Beickman K, Otte E, Rymond BC. Progression through the spliceosome cycle requires Prp38p function for U4/U6 snRNA dissociation. EMBO J. 1998;17(10):2938–46.View ArticlePubMedPubMed CentralGoogle Scholar
- Rechsteiner M, Rogers SW. PEST sequences and regulation by proteolysis. Trends Biochem Sci. 1996;21(7):267–71.View ArticlePubMedGoogle Scholar
- epestfind. http://emboss.bioinformatics.nl/cgi-bin/emboss/epestfind. Accessed 01 Nov 2016.
- Ward JJ, Sodhi JS, McGuffin LJ, Buxton BF, Jones DT. Prediction and functional analysis of native disorder in proteins from the three kingdoms of life. J Mol Biol. 2004;337(3):635–45.View ArticlePubMedGoogle Scholar
- Pace CN, Scholtz JM. A helix propensity scale based on experimental studies of peptides and proteins. Biophys J. 1998;75(1):422–7.View ArticlePubMedPubMed CentralGoogle Scholar
- Coelho Ribeiro Mde L, Espinosa J, Islam S, Martinez O, Thanki JJ, Mazariegos S, Nguyen T, Larina M, Xue B, Uversky VN. Malleable ribonucleoprotein machine: protein intrinsic disorder in the Saccharomyces cerevisiae spliceosome. Peer J. 2013;1:e2.View ArticlePubMedGoogle Scholar
- Korneta I, Bujnicki JM. Intrinsic disorder in the human spliceosomal proteome. PLoS Comput Biol. 2012;8(8), e1002641.View ArticlePubMedPubMed CentralGoogle Scholar
- Deckert J, Hartmuth K, Boehringer D, Behzadnia N, Will CL, Kastner B, Stark H, Urlaub H, Luhrmann R. Protein composition and electron microscopy structure of affinity-purified human spliceosomal B complexes isolated under physiological conditions. Mol Cell Biol. 2006;26(14):5528–43.View ArticlePubMedPubMed CentralGoogle Scholar
- Edgar RC. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 2004;32(5):1792–7.View ArticlePubMedPubMed CentralGoogle Scholar
- Waterhouse AM, Procter JB, Martin DM, Clamp M, Barton GJ. Jalview Version 2--a multiple sequence alignment editor and analysis workbench. Bioinformatics. 2009;25(9):1189–91.View ArticlePubMedPubMed CentralGoogle Scholar
- Rice P, Longden I, Bleasby A. EMBOSS: the European Molecular Biology Open Software Suite. Trends Genet. 2000;16(6):276–7.View ArticlePubMedGoogle Scholar
- Rost B, Yachdav G, Liu J. The predict protein server. Nucleic Acids Res. 2004;32(Web Server issue):W321–6.View ArticlePubMedPubMed CentralGoogle Scholar
- Ulrich A, Andersen KR, Schwartz TU. Exponential megapriming PCR (EMP) cloning--seamless DNA insertion into any target plasmid without sequence constraints. PLoS One. 2012;7(12):e53360.View ArticlePubMedPubMed CentralGoogle Scholar
- Studier FW. Protein production by auto-induction in high density shaking cultures. Protein Expr Purif. 2005;41(1):207–34.View ArticlePubMedGoogle Scholar