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. 2006 Jan;16(1):66–77. doi: 10.1101/gr.3936206

Systematic genome-wide annotation of spliceosomal proteins reveals differential gene family expansion

Nuno L Barbosa-Morais 1,2, Maria Carmo-Fonseca 2, Samuel Aparício 1,3,4
PMCID: PMC1356130  PMID: 16344558

Abstract

Although more than 200 human spliceosomal and splicing-associated proteins are known, the evolution of the splicing machinery has not been studied extensively. The recent near-complete sequencing and annotation of distant vertebrate and chordate genomes provides the opportunity for an exhaustive comparative analysis of splicing factors across eukaryotes. We describe here our semiautomated computational pipeline to identify and annotate splicing factors in representative species of eukaryotes. We focused on protein families whose role in splicing is confirmed by experimental evidence. We visually inspected 1894 proteins and manually curated 224 of them. Our analysis shows a general conservation of the core spliceosomal proteins across the eukaryotic lineage, contrasting with selective expansions of protein families known to play a role in the regulation of splicing, most notably of SR proteins in metazoans and of heterogeneous nuclear ribonucleoproteins (hnRNP) in vertebrates. We also observed vertebrate-specific expansion of the CLK and SRPK kinases (which phosphorylate SR proteins), and the CUG-BP/CELF family of splicing regulators. Furthermore, we report several intronless genes amongst splicing proteins in mammals, suggesting that retrotransposition contributed to the complexity of the mammalian splicing apparatus.

In most eukaryotes, functional messenger RNAs (mRNAs) are produced by accurately removing noncoding sequences (introns) from precursors (pre-mRNAs) in a process termed “RNA splicing.” The spliceosome, a large multicomponent ribonucleoprotein complex, carries out this intron excision (Burge et al. 1999; Jurica and Moore 2003). Extensive genetic and biochemical studies in a variety of systems have revealed that the spliceosome contains five essential small RNAs (snRNAs), each of which functions as an RNA-protein complex called a small nuclear ribonucleoprotein (snRNP). Each snRNP comprises one of these five snRNAs bound stably to two classes of proteins, i.e., Sm proteins, which are present in all snRNPs, and specific proteins that are uniquely associated with only one snRNP (Luhrmann et al. 1990). Higher eukaryotes have two distinct types of spliceosomes. The major or U2-type spliceosome, which catalyzes the removal of most introns, is composed of U1, U2, U4, U5, and U6 snRNPs. The minor or U12-type spliceosome, which recognizes <1% of all human introns, comprises U11, U12, U4atac, U5, and U6atac snRNPs (Patel and Steitz 2003). In addition to snRNPs, splicing requires many non-snRNP protein factors. Recent improved methods to purify spliceosomes coupled with advances in mass spectrometry have revealed that the spliceosome may be composed of as many as 300 distinct proteins (Jurica and Moore 2003; Nilsen 2003).

The initial events of spliceosome assembly require recognition of specific sequences located at the 5′ and 3′ splice sites, which define the intron boundaries. In metazoans, however, the splice site sequences are only weakly conserved and although introns are excised with a high degree of precision, at least 74% of human genes encode alternatively spliced mRNAs (Johnson et al. 2003). Alternative splicing is the process by which multiple mRNAs can be generated from the same pre-mRNA by the differential joining of 5′ and 3′ splice sites. Alternative splicing produces multiple mRNAs encoding distinct proteins, thus expanding the coding capacity of genes and contributing to the proteomic complexity of higher organisms (Brett et al. 2002; Maniatis and Tasic 2002; Black 2003).

In general, alternative splicing is regulated by protein factors that recognize and associate with specific RNA sequence elements either to enhance or to repress the ability of the spliceosome to recognize and select nearby splice sites (Smith and Valcarcel 2000; Maniatis and Tasic 2002). The multiplicity of protein-protein and protein-RNA interactions that modulate the association of the spliceosome with the pre-mRNA is thought to control alternative splicing (Caceres and Kornblihtt 2002; Graveley 2002; Black 2003).

The evolutionary history of the splicing machinery has not been fully elucidated, in part because appropriate near-complete genome sequences have only recently become available. The recent sequencing and annotation of the genomes of the Japanese puffer fish, Fugu rubripes (Aparicio et al. 2002), and the sea squirt, Ciona intestinalis (Dehal et al. 2002), allows us now to fill that gap with fiducial branches of distant vertebrates and chordates, respectively, providing an opportunity to exhaustively look at splicing factors in those species and extend our knowledge about their evolution. In this study, we report a semiautomated computational pipeline designed to identify and annotate splicing factors in representative species of eukaryotes.

Results

Pipeline-assisted annotation of splicing factors

Although recent reports have identified up to 300 distinct proteins associated with the spliceosome (Rappsilber et al. 2002; Zhou et al. 2002), many of these new proteins have not yet been shown to function in splicing and, therefore, they cannot be considered as bona fide splicing factors (Jurica and Moore 2003). In this study, we limited our analysis to proteins for which there is experimental evidence of their involvement in splicing. Our first goal was to enumerate and annotate the genes encoding spliceosomal proteins in the genomes of human, pufferfish, Ciona, the budding yeast Saccharomyces cerevisiae, the fission yeast Schizosaccharomyces pombe, the plant Arabidopsis thaliana, and several species of archaebacteria and protozoa (see Methods; Fig. 1; Supplemental Table S1).

Figure 1.

Figure 1.

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Schematics of the computational pipeline flow. Sources, software, and parameters are represented in blue and species in red.

Although the availability of “raw” and “first pass annotated” genomes (for example the ones in Ensembl [Hubbard et al. 2002]) is proving indispensable for genome-wide studies, detailed analyses are still hampered by the fact that most databases are “contaminated” with erroneous annotation. In many cases, the current algorithms used in completely automated gene-building pipelines unreliably predict features such as short exons. The algorithms are particularly ineffective with repetitive protein motifs, such as those in RS (arginine-serine-rich) domains, responsible for the protein-protein interactions of SR (Ser-Arg) proteins (important splicing factors—see below). The goal of our semiautomated pipeline was to search ab-initio the raw genomic sequence of representative eukaryotes and thus to complement pre-existing annotations, even though these acted as a seed for the pipeline. This approach demanded manual inspection and validation of the results. We therefore visually inspected a total of 1894 putative spliceosomal proteins across eukaryotic genomes, and we manually curated 224 sequences (12%). The results are listed in Supplemental Table S2. Despite the effort made to manually correct sequences, errors and uncertainties remain, especially for genes poorly supported with EST evidence, and this reduces the precision of the phylogenetic analysis (namely for Parsimony methods) and the consistency of tree topology between different methods of phylogenetic inference (all of the trees can be found in the Supplemental materials). We were unable to correct completely 388 proteins (20%) of ambiguous sequence. We identified only five putative splicing factors (all from Fugu) that had no previously annotated gene locus. We also report three factors (from Zebrafish) that were annotated in older versions of Ensembl, but do not appear in version 30. In the process of manual curation, we have identified 83 putative pseudo-genes that Ensembl annotates as active genes in human and mouse (Supplemental Table S3; see below), indicating that automated annotation is oversensitive.

Selective expansion of splicing regulatory protein families

Having enumerated all currently known splicing proteins, we asked whether major patterns of protein family expansion were evident between different animal phyla. We looked at the genes encoding the seven Sm protein families that associate with all the snRNAs, the Lsm protein families that associate with the U6 snRNA, and several snRNP-specific protein families. Most of these spliceosomal components show apparent one:one orthology mapping (or numerical concordance in the occurrence of paralogs) between vertebrates, invertebrates, and unicellular eukaryotes, consistent with previous reports (see Will and Luhrmann 2001). In contrast, we observed a different evolutionary pattern of the minor spliceosome U11/U12-snRNP proteins; they are absent from protozoa, trypanosomes, yeasts, and the nematode worm Caenorhabditis elegans (Table 1), but present in Arabidopsis, consistent with the identification of U12-dependent introns in this plant (Zhu and Brendel 2003).

Table 1.

Compilation of U11/U12 snRNP and DExD/H-box (DEAD) proteins identified in the analyzed genomes

Family Human Mouse Rat Chicken Fugu Zebrafish Tetraodon Ciona
U11/U12-20 UB20 UB20 UB20 UB20 UB20 UB20a UB20
UB20b
U11/U12-25 UB25 UB25 UB25 UB25 UB25 UB25 UB25
U11/U12–31 UB31 UB31 UB31 UB31 UB31 UB31 UB31 UB31
U11/U12-35 UB35 UB35 UB35 UB35 UB35 UB35 UB35
U11/U12-48 UB48 UB48 UB48 UB48 UB48 UB48 UB48 UB48
U11/U12-65 UB65 UB65 UB65 UB65 UB65 UB65 UB65 UB65
ABS ABS ABS ABS ABS ABS ABS ABS ABS
DDX26 DDX26 DDX26 DDX26 DDX26 DDX26 DDX26 DDX26 DDX26
DX26b
DD26B DD26B DD26B DD26B DD26B DD26B
DDX39 DDX39 DDX39 DDX39 DDX39 DDX39 DDX39
BAT1 BAT1 BAT1 BAT1 BAT1
DDX3XY DDX3X DDX3X DDX3X DDX3 DDX3a DDX3 DDX3 DDX3
DDX3Y DDX3Y DDX3b
DDX46 DDX46 DDX46 DDX46 DDX46 DDX46 DDX46 DDX46 DDX46
DDX48 DDX48 DDX48 DDX48 DDX48 DDX48 DDX48 DDX48 DDX48
DHX15 DHX15 DHX15 DHX15 DHX15 DHX15 DHX15 DHX15 DHX15
DHX16 DHX16 DHX16 DHX16 DHX16 DHX16 DHX16 DHX16 DHX16
DHX35 DHX35 DHX35 DHX35 DHX35 DHX35 DHX35 DHX35
DHX38 DHX38 DHX38 DHX38 DHX38 DHX38 DHX38 DHX38 DHX38
DHX8 DHX8 DHX8 DHX8 DHX8a DHX8a DHX8a DHX8 DHX8
DHX8b DHX8b DHX8b
DHX9 DHX9 DHX9 DHX9 DHX9 DHX9 DHX9a DHX9
DHX9b
KIAA0052 K052 K052 K052 K052 K052 K052 K052 K052
P68p72 DDX5 DDX5 DDX5 DDX5 DDX5 DDX5 DDX5 p68
DDX17 DDX17 DDX17 DDX17 DD17a DDX17 DDX17
DD17b
U5-100a DDX23 DDX23 DDX23 DDX23 DDX23 DDX23
U5-200a U5200 U5200 U5200 U5200 U5200 U5200 U5200 U5200
Family Fly Mosquito C. elegans Arabidopsis S. pombe S. cerevisiae Plasmodium T. cruzi
U11/U12-20 UB20
U11/U12-25 UB25a
UB25b
U11/U12–31 UB31 UB31
U11/U12-35 UB35 UB35
U11/U12-48
U11/U12-65 UB65 UB65
ABS ABS ABS ABS ABSa ABS
ABSb
DDX26 DDX26 DDX26
DDX39 WM6 DDX39 DDX39 DDX39 UAP56 SUB2 DDX39 DDX39
DDX3XY DDX3 DDX3 DDX3a DDX3a DED1 DED1 DDX3a
DDX3b DDX3b DBP1 DDX3b
DDX3c DDX3c
DDX3d
DDX46 DDX46 DDX46 DD46a DD46a PRP11 PRP5 DDX46 DDX46
DD46b DD46b
DDX48 DDX48 DDX48 DD48a IF4Aa EIF4A FAL1 EIF DDX48
DD48b IF4Ab
DHX15 DHX15 DHX15 DHX15 DH15a DHX15 PRP43 DHX15 DH15a
DH15b DH15b
DH15c
DHX16 DHX16 DHX16 DH16a CDC28
DH16b
DHX35 DHX35 DHX35 DHX35 DHX35
DHX38 DHX38 DHX38 DHX38 DHX38 PRP16 PRP16 DHX38 DHX38
DHX8 DHX8 DHX8 DHX8 DHX8 DHX8 PRP22 DHX8 DHX8
DHX9 MLE DHX9 DHX9
KIAA0052 K052 K052 K052 K052a K052a MTR4 K052 K052a
K052b K052b K052b
P68p72 DDXP DDXP DDXP RH20 DBP2 DBP2 DDXP DDXPa
RH30 DDXPb
DDXPc
U5-100a DDX23 DDX23 DDX23 DDX23 PRP28 PRP28 DDX23
U5-200a U5200 U5200 U5200 U520a BRR2 BRR2 U5200 U520a
U5Hyp U520b U520b
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Detailed identification of each gene is provided in Table S2. Small termination characters identify species/phylum specific duplications.

a

Families annotated as snRNP specific

In addition to snRNPs, the spliceosome comprises many non-snRNP protein factors, including DExD/H-box proteins, SR proteins, and hnRNP proteins. DExD/H-box proteins constitute a prominent family of core splicing factors. Genetic studies in S. cerevisiae have implicated eight DExD/H-box proteins in splicing (Staley and Guthrie 1998). Each of these conserved proteins (Prp2p, Prp16p, Prp22p, Prp43p, Brr2, Prp5p, Prp28p, Sub2p) is required for pre-mRNA splicing. Seven additional DExD/H-box proteins were recently found associated with mammalian spliceosomes (Jurica and Moore 2003). As shown in Table 1, no major expansion of the DExD/H-box gene family occurred during evolution.

The SR proteins, characterized by their typical RS domain containing repeated Arg/Ser dipeptides, are essential factors required for both constitutive and alternative splicing (Maniatis and Tasic 2002). Our results show that metazoans contain nine families of SR proteins, six of which have two or more members in mammals, whereas in unicellular eukaryotes there are only one or two SR protein genes (Table 2). Thus, the diversity of SR proteins seems to have emerged with multicellularity. Consistent with previous reports, we found no SR proteins in budding yeast, but two proteins in fission yeast (Tacke and Manley 1999; Kaufer and Potashkin 2000), and we confirmed the existence of 19 SR protein genes in Arabidopsis (Kalyna and Barta 2004; Reddy 2004).

Table 2.

Compilation of SR proteins identified in the analyzed genomes

Family Human Mouse Rat Chicken Fugu Zebrafish Tetraodon Ciona Fly Mosquito C. elegans Arabidopsis S. pombe Plasmodium T. cruzi
9G8-SRp20 9G8 9G8 9G8 9G8a 9G8 9G8a 9G8 9G8a 9G8 9G8 RSP6 RS21
SR20 SR20 SR20 9G8b SR20a 9G8b SR20 9G8b RBP1 RBP1 RSPY RS22
SR20 SR20b 9G8c SR20 RBP1L RSF1 RS22A
SR20a RSF1 RS32a
SR20b RS33a
p54 p54 p54 p54 p54 p54a p54 p54a SR86a p54 p54 p54
SR86 SR86 SR86 SR86 p54b SR86 p54b SR86b
SR86 p54c
RY1 RY1 RY1 RY1 RY1 RY1 RY1 RY1 RY1 RY1 RY1 RY1
SC35 SC35 SC35 SC35 SC35 SC35a SC35a SC35 SC35 SC35 SC35 SC28a SRP1b
SR46 SC35b SC35b SC30a
SC30Aa
SC33a
SC35
SRm300 SR300 SR300 SR300 SR300 SR300 SR300 SR300 SR300 SR300 SRRM2 SR45 SFb
SRp30c-ASF ASF ASF ASF ASF ASFa ASFa ASF ASFa SF2 SF2 SF2 RS31Aa
SR30C SR30C SR30C ASFb ASFb ASFb SR34
SR30C SR30C SR34A
SR34B
SRp30
SRp40-55-75 SR40 SR40 SR40 SR40a SR40a SR40a SR40a SR40a SR55 SR40 RSP1 RSp31a SRP2b SR1b
SR55 SR55 SR55 SR40b SR40b SR40b SR40b SR40b RSP2 RSp40a
SR75 SR75 SR75 SR55 SR55 SR55a SR40c RSP5 RSP41a
SR75 SR75 SR55b SR55
SR75
Topol-B T1B T1B T1B T1B T1Ba T1Ba T1Ba T1B T1B
T1Bb T1Bb T1Bb
Tra2 Tra2A Tra2A Tra2A Tra2A Tra2B Tra2A Tra2B Tra2 Tra2 Tra2a Tra2
Tra2B Tra2B Tra2B Tra2B Tra2B Tra2b
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Detailed identification of each gene is provided in Table S2. Small termination characters identify species/phylum specific duplications. None of the analyzed SR protein genes was found for Saccharomyces cerevisiae.

a

Arabidopsis-specific SR proteins, technically considered orthologs of the human proteins in the same family (reciprocal BLAST hit) but exhibiting a considerably lower degree of identity with the human factor than their Arabidopsis paralogs

b

SR proteins in unicellular eukaryotes can be considered common homologs of all the SR proteins in metazoans; here we include them in the same families of their technical human orthologs (reciprocal BLAST hit)

The hnRNP proteins are a large group of molecules identified by their association with unspliced mRNA precursors (hnRNAs). The hnRNP proteins A, C, F, G, H, I (also termed PTB), and M have been implicated in the regulation of splicing (Black 2003). We find that a single S. pombe protein shows significant sequence homology to hnRNPs, whereas 13 gene families are found in metazoans (Table 3). For each invertebrate hnRNP in Ciona, insects, or worms, there are, on average, three co-orthologs in the vertebrates human, mouse, and Fugu. Ciona has 16 hnRNP genes, whereas human has 37. Thus, a striking expansion of hnRNP protein gene families occurred in vertebrates.

Table 3.

Compilation of hnRNP proteins identified in the analyzed genomes

Family Human Mouse Rat Chicken Fugu Zebrafish Tetraodon Ciona Fly Mosquito C. elegans Arabidopsis S. pombe T. cruzia
hnRNP-A ROA0 ROA0 ROA1 ROA0 ROA0 ROA0a ROA0 ROA1a RO87F RO87F ROAa ROAa
ROA1 ROA1 ROA2 ROA1 ROA1 ROA0b ROA1 ROA1b RO97D ROAb ROAb
ROA2 ROA2 ROA3 ROA3 ROA0c ROA3 ROA3 ROA1 ROAc
ROA3 ROA3 ROA3
hnRNP-C RLY RLY RLY RLY RLYLa RLYa RLYLa ROC
RLYL RLYL RLYL RLYL RLYLb RLYb RLYLb
ROC ROC ROC ROCa RLYLa ROC
ROCL ROCb RLYLb
ROCa
ROCb
hnRNP-D-U2 ROAB ROAB ROAB ROABa ROABa ROAB ROABa ROAB RO40 RO40 RODU2 RODa?
ROD0 ROD ROD0 ROABb ROABb ROD0 ROABb ROD RODb?
RODL ROD0 RODL RODL ROD0 RODL ROD0
RODL
hnRNP-E PCB1 PCB1 PCB1 PCB3 PCB2a PCB2 PCB2 PCB PCB PCB PCB
PCB2 PCB2 PCB2 PCB2b PCB3 PCB3a
PCB3 PCB3 PCB3 PCB3a PCB3b
PCB4 PCB4 PCB4 PCB3b
PCB4a
PCB4b
hnRNP-F-H GRSF1 GRSF1 GRSF1 GRSF1 GRSF1 GRSF1 GRSF1 ROFHa ROFH ROFH ROFHa ROFHa ROFHa?
ROF ROF ROF ROH1 ROFH ROFHa ROFH ROFHb ROFHb ROFHb ROFHb?
ROH1 ROH1 ROH1 ROH3 ROH3 ROFHb ROH3
ROH2 ROH2 ROH2 ROH3
ROH3 ROH3 ROH3
hnRNP-G ROG ROG ROG ROG ROG ROG ROG
ROGT ROGT ROGT
hnRNP-I PTB1 PTB1 PTB1 PTB1 PTB1a PTB1a PTB1 PTBa PTB PTB PTB PTBa PTBa?
PTB2 PTB2 PTB2 PTB2 PTB1b PTB1b PTB2 PTBb PTBb PTBb?
ROD1 ROD1 ROD1 ROD1 PTB2 PTB2a ROD1 PTBc PTBc?
smPTB smPTB ROD1 PTB2b
ROD1
hnRNP-K ROK ROK ROK ROKa ROKa ROKa ROK ROK ROK ROK
ROKb ROKb ROKb
hnRNP-L ROL ROL ROL ROL ROLa ROL ROL ROL ROL
ROLH ROLH ROLH ROLHa ROLb ROL
ROLHb ROLH
hnRNP-M Myel Myel Myel Myel Myel Myel Myel ROM ROM ROM ROM ROM?
ROM ROM ROM ROM ROM ROM ROM
hnRNP-R ROQ ROQ ROQ ROQ ROQa ROQ ROQa ROQR ROQR ROQR ROQR ROQRa
ROR ROR ROR ROR ROQb ROR ROQb ROQRb
ROR ROR ROQRc
hnRNP-U E1BA E1BA E1BA E1BA E1BAa E1BA ROUa ROU ROU ROU
ROU ROU ROU ROU E1BAb ROU ROUb
ROUHY ROUHY ROUHY ROUH ROU0 ROUHY
ROUa
ROUb
Musashi MUS1 MUS1 MUS1 MUS1 MUS1 MUS1 MUS1 MUSa MUS MUS MUS
MUS2 MUS2 MUS2 MUS2 MUS2a MUSb
MUS2b
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Detailed identification of each gene is provided in Table S2. Small termination characters identify species/phylum specific duplications. None of the analyzed hnRNP genes was found for Saccharomyces cerevisiae and Plasmodium falciparum.

a

Proteins signed with `?' are technically orthologs (reciprocal BLAST hit) but the large evolutionary distance (and low sequence similarity) and the absence of experimental data does not allow us to classify them as functional homologs

Interestingly, gene families encoding additional splicing regulators have also expanded during the evolution of primitive metazoans into vertebrates (Table 4). These include the CLK (CDC-like) and SRPK (SR-protein-specific) kinases that phosphorylate SR proteins, modulating their function in splicing; the CUGBP (CUG-binding) and ETR-like proteins (CELF) implicated in tissue-specific and developmentally regulated alternative splicing and the alternative splicing regulators FUSE (far upstream element binding), and Elav (embryonic lethal abnormal visual) proteins (for a recent review see Black 2003).

Table 4.

Evolution of miscellaneous splicing regulatory proteins

Family Human Mouse Rat Chicken Fugu Zebrafish Tetraodon Ciona Fly Mosquito C. elegans Arabidopsis S. pombe S. cerevisiae Plasmodium T. cruzi
CLK CLK1 CLK1 CLK1 CLK2 CLK2a CLK2 CLK2a CLK CLK CLK CLK CLK CLK CLK CLK CLKa
CLK2 CLK2 CLK2 CLK3 CLK2b CLK4 CLK2b AFC1 CLKb
CLK3 CLK3 CLK3 CLK4 CLK4 CLK4 AFC2
CLK4 CLK4 CLK4 AFC3
CUG CUG1 CUG1 CUG1 CUG1 CUG1 CUG1 CUG2a CUG1 CUGa CUGa CUG RBPa
CUG2 CUG2 CUG2 CUG2 CUG2a CUG2 CUG2b ETR3 CUGb CUGb RBPb
CUG3 CUG3 CUG3 CUG3 CUG2b CUG3 CUG3a
CUG4 CUG4 CUG4 CUG4 CUG3a CUG4 CUG3b
CUG5 CUG5 CUG5 CUG5 CUG3b CUG5 CUG4
CUG6 CUG6 CUG6 CUG6 CUG4 CUG5
CUG5
ELAV ELAV1 ELAV1 ELAV1 ELAV1 ELV1a ELV1a ELAV1 ELAVa ELAVa ELAVa ELAV
ELAV2 ELAV2 ELAV2 ELAV3 ELV1b ELV1b ELAV2 ELAVb ELAVb ELAVb
ELAV3 ELAV3 ELAV3 ELAV4 ELAV3 ELAV2 ELAV3 ELAVc ELAVc
ELAV4 ELAV4 ELAV4 ELAV4 ELAV3 ELAV4
ELAV ELAV4
ELAV
FUSE FUSE1 FUSE1 FUSE2 FUSE1 FUSE1 FUSE1 FUSE1 FUSE PSI PSI FUSEa KHa
FUSE2 FUSE2 FUSE3 FUSE2 FUSE2 FUS2a FUSE2 FUSEb KHb
FUSE3 FUSE3 FUSE3 FUSE3 FUS2b FUS3a FUSEc KHc
FUSE3 FUS3b FUSEd
FUSEe
SRPK MSSK1 MSSK1 MSSK1 SRPK1 MSSK1 MSSK1 SRK1a SRPK SRPK SRPK SRPK MSSKa SRPK SRPK SRPK SRPK
SRPK1 SRPK1 SRPK1 SRPK2 SRPK1 SRK1a SRK1b MSSKb
SRPK2 SRPK2 SRPK2 SRPK SRPK2 SRK1b SRK1c MSSKc
SRPKa SRPK2 SRPK2 SRPKa
SRPKb SRPKa SRPKa SRPKb
SRPKb SRPKb
SRPKc SRPKc
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Detailed identification of each gene is provided in Table S2. Small termination characters identify species/phylum specific duplications.

Since genome duplication is known to have occurred at the vertebrate stem (Mazet and Shimeld 2002; McLysaght et al. 2002), we performed a phylogenetic analysis, using rate-linearized trees (see Methods) to determine whether the splicing factor family expansions are coincident with that duplication. Despite some topological inconsistencies between the different methods of phylogenetic inference, the evolutionary trees we generated are most consistent with the model that hnRNP genes underwent one or two rounds of duplication just after the divergence of vertebrates (Fig. 2A,B) and urochordates.

Figure 2.

Figure 2.

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Evolutionary relationship among the protein members of hnRNP F/H family in several eukaryotes, i.e., human (Hsap), mouse (Mmus), rat (Rnor), chicken (Ggal), Fugu (Frub), zebrafish (Drer), Tetraodon (Tnig), Ciona intestinalis (Cint), fruit fly (Dmel), mosquito (Agam), C. elegans (Cele), Arabidopsis (Atha), and Trypanosoma (Tcru). Vertebrate factors are highlighted in blue, red, and shades of green. (A) Rooted Neighbor-Joining phylogenetic tree generated using ClustalW (1000 bootstraps), based on amino-acid alignment generated by T-Coffee. Bootstrap values are shown. Branch lengths are scaled in arbitrary units. (B) Rooted Gamma-corrected Maximum-Likelihood phylogenetic tree generated using GAMMA and the Phylip program Proml, based on amino-acid alignment generated by T-Coffee. Branch lengths are scaled in arbitrary units.

Furthermore, analysis of the teleost radiation, and of Arabidopsis revealed several localized gene duplications in Fugu, the zebrafish Danio rerio, Tetraodon (all teleosts), and Arabidopsis. These results are consistent with the currently accepted models proposing additional rounds of whole-genome duplication in ray-finned and lobe-finned fish, before teleost radiation (Amores et al. 1998; Aparicio et al. 2002; Christoffels et al. 2004), and the propensity of angiosperms to become polyploid (Simillion et al. 2002; Bowers et al. 2003). Thus, teleost fish and plants tend to have more copies of splicing genes than do mammals (Tables 1, 2, 3, 4). However, there is no evidence for additional selective expansion of any particular family of splicing proteins in these organisms, beyond that which had occurred in the stem organism.

The domain evolution of splicing factors

Our data show conservation of the protein domain structure of splicing factors across species, and we found no evidence for domain shuffling. We observed no trend for gain or loss of domains in families of splicing factors, as has occurred in other nuclear protein families (for example, in the Polycomb and Trithorax protein families) (Ringrose and Paro 2004). We checked, for example, whether the expansion of SR protein families coincided with the appending of RS domains onto general RNA-binding splicing factors. In species without SR proteins, we found no relevant homology with SR protein RNA recognition motifs (RRMs). Each factor seems to have evolved as a whole and its domains have evolved together (Fig. 3). Similarly, for the hnRNP families that are expanded in vertebrates, the motif structures are generally conserved (Fig. 4). One exception is hnRNP H3, which in mammals and chicken appears to have lost the first of the three RRM's that are common to its paralogs.

Figure 3.

Figure 3.

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Evolutionary relationship among the RNA-recognition motifs (RRM) of members of the family SRp30c-ASF for several eukaryotes, i.e., human (Hsap), mouse (Mmus), chicken (Ggal), Fugu (Frub), Ciona (Cint), fruit fly (Dmel), C. elegans (Cele), Arabidopsis (Atha), and Plasmodium (Pfal) (for simplicity only one rodent, one teleost, and one insect are shown). Amino-acid positions of each domain within the protein are also indicated in the domain identification. The unrooted Neighbor-Joining phylogenetic tree was generated using ClustalW (1000 bootstraps) based on amino-acid alignment generated by T-Coffee. Bootstrap values are shown. Branch lengths are scaled in arbitrary units. RRM1 in Ggal_ASF and Cint_ASFb corresponds to RRM2 in the other proteins as their sequences are truncated in the N-terminal. Pfal_SF is found to have only one RRM. Atha_RS31A can be technically considered an ortholog of the Hsap_SR30C (reciprocal BLAST hit) but exhibits a considerably lower degree of identity (36%) with the human factor than its Arabidopsis paralogs (e.g., 53% for Atha_SRp30).

Figure 4.

Figure 4.

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Evolutionary relationship among the RNA-binding K-Homology (KH) domains of members of the family hnRNP-E/PCB for several metazoans, i.e., human (Hsap), mouse (Mmus), chicken (Ggal), Fugu (Frub), Ciona (Cint), fruit fly (Dmel), and C. elegans (Cele) (for simplicity only one rodent, one teleost, and one insect are shown). Amino-acid positions of each domain within the protein are also indicated in the domain identification. The unrooted Neighbor-Joining phylogenetic tree was generated using ClustalW (1000 bootstraps) based on amino-acid alignment generated by T-Coffee. Bootstrap values are shown. Branch lengths are scaled in arbitrary units.

Retrotransposition and identification of putative novel splicing factors and pseudogenes in mammals

The absence of introns from mammalian genes is often indicative of retrotransposition, where a spliced mRNA is reverse transcribed into DNA and integrates back into the genome. Retrotransposition appears to have contributed as a general mechanism of gene duplication amongst mammals. We found that, with the exception of U2AF26 (a mammalian splicing factor [Shepard et al. 2002] that diverged from U2AF35 before vertebrates' radiation and is likely to have been lost by defunctionalization in teleosts) and Sm N, all of the mammalian specific factors SRp46, U2AF1-RS1 and hnRNPs C-like, E1, smPTB and G-T are intronless, whereas their closer paralogs are multiexonic. SRp46, U2AF1-RS1, hnRNP E1, and hnRNP G-T have previously been reported to be retrotransposons (Soret et al. 1998; Makeyev et al. 1999; Elliott et al. 2000; Wang et al. 2004), which is consistent with our data. We therefore propose that retrotransposition contributed to generate the diversity of the splicing machinery observed in mammals.

We found evidence for an additional seven mouse putative intronless genes that appear to have no frame disruption in their coding sequences and for which we find evidence for transcription (Supplemental Table S4) and/or have an outstandingly high ratio of synonymous/nonsynonymous substitutions when compared with the closest active paralogue. Six of these putative intronless genes are annotated in Ensembl, but one of the genes is located in an unannotated genomic region. Two putative intronless genes exhibit transcript sequences equal to their closest paralogs. Whether these are novel functional splicing genes in mouse or very recent pseudogenes remains an open question.

In addition, we identified 107 human and 90 mouse putative pseudogenes (Supplemental Tables S3 and S5), none being found in other phyla. Of these, 30 human and 53 mouse pseudogenes are annotated as putative functional genes in Ensembl (Table S3). The majority (∼80%) of all the analyzed intronless genes/pseudogenes contain evidence for surrounding LINE1 or LTR (long terminal repeat) sequences (repeats associated with transposable elements [Kazazian Jr. 2004]) and are therefore likely to be retrotransposons. Some families of Sm proteins and the hnRNP-A family contain particularly large numbers of retrotransposons (Supplemental Tables S3 and S5).

Discussion

Here we report a systematic comparison of the genes encoding the splicing machinery across diverse phyla. We designed a semiautomated computational pipeline to identify and annotate spliceosomal proteins that will also assist in the rapid reannotation of new splicing proteins as genomic sequences are updated. Our analysis shows differential gene family expansions across the eukaryotic lineage, with a disproportionate expansion of hnRNP proteins in vertebrates.

Although the origin of introns remains unknown, current data strongly indicate that introns and a spliceosome sufficient for their excision were present in the last common ancestor of eukaryotes (Johnson 2002; Collins and Penny 2005). Introns have been discovered in eukaryotes as primitive as the single-celled parasite Giardia lamblia (Nixon et al. 2002) and its close relative Carpendiemona membranifera (Simpson et al. 2002), and a core spliceosomal protein gene (Prp8) is remarkably conserved between metazoans and the deep-branching protist Trichomonas vaginalis (Fast and Doolittle 1999). Our finding that genes encoding snRNP proteins are generally conserved in animals, Arabidopsis, yeasts, trypanosomes, and Plasmodium is consistent with previous reports (for review, see Will and Luhrmann 2001). Our observation that Plasmodium, trypanosomes, yeasts, and C. elegans lack U11/U12 protein homologs is also in agreement with the hypothesis that the minor (U12-dependent) spliceosome was absent from the “first eukaryote” (Collins and Penny 2005).

In contrast to the conservation of snRNP protein genes, our analysis reveals that metazoans have many more genes implicated in the regulation of splicing than unicellular eukaryotes. Most probably, splicing regulatory proteins evolved as a consequence of whole-genome duplications that occurred at the vertebrate stem (Mazet and Shimeld 2002; McLysaght et al. 2002). According to the “classical” model for selective retention of gene family duplicates (Ohno 1970; Force et al. 1999; Nei and Rooney 2004), one of the duplicate genes retained the original function, while the other accumulated mutations that eventually conferred an advantageous new function (neofunctionalization).

We provide surprising evidence that retrotransposition introduced an additional level of diversity to the mammalian splicing machinery. Despite the fact that the majority of retrotransposons are nonfunctional (Goncalves et al. 2000), and that intronless genes may be transcribed less efficiently than their intron-containing homologs (Le Hir et al. 2003), we identified several retrotransposed genes, specific to mammals, encoding multifunctional RNA-binding proteins. These include SRp46 (Soret et al. 1998), hnRNP E1 (Leffers et al. 1995; Ostareck-Lederer et al. 1998; Krecic and Swanson 1999; Reimann et al. 2002; Bandiera et al. 2003; Persson et al. 2003; Antony et al. 2004; de Hoog et al. 2004; Morris et al. 2004), hnRNP G-T (Elliott et al. 2000; Nasim et al. 2003), smPTB (Gooding et al. 2003), and U2AF1-RS1 (Wang et al. 2004). We also identified seven mouse putative novel active retrotransposed genes, paralogs of NHP2-like, U1C, LSm6, LSm7, SmD2, SmG, and U2AF35.

Although splicing of introns from pre-mRNAs occurs in practically all eukaryotes, alternative splicing is important and widespread only in multicellular organisms. The yeast S. cerevisiae has introns in only ∼3% of its genes and only six genes with more than one intron (Barrass and Beggs 2003). Although in the fission yeast S. pombe, 43% of the genes are spliced, with many of them containing multiple introns (Wood et al. 2002), no regulated alternative splicing has been detected in this organism or in any other unicellular eukaryote (Barrass and Beggs 2003; Ast 2004).

There are two current models to explain the evolution of alternative splicing, which are not mutually exclusive (Ast 2004). One is based on the accumulation of mutations that make splice sites suboptimal (or “weaker”), providing an opportunity for the splicing machinery to skip that site. In the second model, the evolution of splicing regulatory factors that either enhance or inhibit the binding of the splicing machinery to constitutive splice sites, it argues, releases the selective pressure from that sequence, resulting in mutations that weaken the splice sites. Our results clearly support this second model, which so far has not received much experimental attention. The choice of splice site is thought to be regulated by altering the binding of the spliceosome to the pre-mRNA. This is achieved by RNA-binding proteins that associate with nonsplice site sequences, located either in exons or introns. The best-characterized families of splicing regulators are SR proteins and hnRNP proteins (for review, see Black 2003). In vitro selection experiments have identified optimal binding sequences for different SR and hnRNP proteins, but the binding sites for a given family member can be fairly degenerate. Moreover, regulatory proteins can act as either splicing activators or repressors, depending on where in the pre-mRNA they bind. We propose, therefore, that the evolution of novel members of splicing regulatory protein families permitted the diversification of their canonical binding sites in pre-mRNAs, giving the cell the potential to produce new transcripts by altering splice choices. This hypothesis may be testable by correlating functional specificity of individual factors for their splice isoforms with the cognate recognition sequences in different species.

Methods

The key steps in our pipeline are illustrated in Figure 1.

All of the human splicing factors (Supplemental Table S6) and homologs annotated for other species were listed and their protein sequences were retrieved. Grouping into families was performed based on full-length homology, functional domains composition, and the Ensembl Protein Family classification (Hubbard et al. 2002) (v30, http://www.ensembl.org). For each family, spurious and truncated proteins were identified and removed manually, and all of the remaining members were aligned with T-Coffee (Notredame et al. 2000) (default parameters). The alignment was used to build a profile HMM (Hidden Markov Model), using HMMER (Eddy 1998) (hmmbuild, hmmcalibrate), with which the proteomes of Fugu, Ciona, and 16 species of Archaea were searched (hmmsearch, e-value = 10-2). In parallel, all of the human splicing factors were BLASTed (Altschul et al. 1990) (tblastn, BLOSUM62 matrix, SEG filter on, e-value = 10-3) against the genomes of Fugu, Ciona, Archaea, Plasmodium, Trypanosomas, and proteomes of the previous species plus A. thaliana, S. pombe, and S. cerevisiae. Gene prediction was carried out in hit unannotated genomic regions, using Wise2 (http://www.ebi.ac.uk/Wise2). A reciprocal BLAST between the protein hits and the human genome and proteome (blastp, BLOSUM62 matrix, SEG filter on, e-value = 10-3) was performed. Gene predictions were again made for hit unannotated genomic regions in Human.

The obtained members of each “complete” family were aligned and a phylogenetic tree was built with ClustalW (Thompson et al. 1994). The families of factors with relevant annotated function benefited from further curation, i.e., removal of false homologs and redundancies, correction of truncated and missannotated proteins, assessment of the likelihood of splice sites. This curation was assisted by BLAST, Wise 2, and EST searches, carried out in the Gene2EST BLAST Server (Gemund et al. 2001) (EMBL, http://woody.embl-heidelberg.de/gene2est) and the NCBI BLAST website (http://www.ncbi.nlm.nih.gov/BLAST, blastn, low complexity filter on), relying on the GenBank/dbEST database (v147.0) (Boguski et al. 1993; Benson et al. 2004).

The same approach was used to identify putative retrotransposons and discriminate pseudo-genes (based on the appearance of frame disruptions like cryptic stop codons and frameshifts introduced by missing or extra nucleotides in the conserved coding region). This procedure was complemented with the estimation of the ratio ds/dn of synonymous/nonsynonymous substitutions (using SNAP—http://www.es.embnet.org/Doc/SNAP/) and the identification of LINEs and LTR elements by searching the involving genomic sequences (1.2 kb upstream and downstream of the putative transcribed sequence) with RepeatMasker (http://www.repeatmasker.org, default parameters).

New alignments were built for the resulting curated families and, for each family, the functional domain composition of its members was compared. The domain organization of proteins relied on the Pfam database (Bateman et al. 2002) (http://www.sanger.ac.uk/Software/Pfam, version 16.0), the HMMER program hmmpfam and the SMART tool (Letunic et al. 2004) (http://smart.embl-heidelberg.de, version 4.0).

We then performed the phylogenetic analysis of all the families by generating bootstrapped Neighbor-Joining (NJ) trees with ClustalW (1000 bootstraps). Alternatively, we bootstrapped our alignments using the Phylip (Felsenstein 1989) program Seqboot (100 bootstraps). Then rooted and bootstrapped NJ and Parsimony trees were built using the Phylip program's Neighbor (preceded by Protdist) and Protpars, respectively. In both cases we generated the consensus trees with Phylip program Consense. We also created rooted Maximum-Likelihood (ML) trees using the Phylip program Proml. For details on tree rooting, see Supplemental Table S7. We did the molecular clock analysis following a procedure similar to that adopted by Christoffels et al. (2004) (Supplemental Table S8).

Supplemental Table S1 summarizes the sources for the whole genomes and predicted proteomes used in our search. The automated searches relied on BioPerl (Stajich et al. 2002) (v1.30, http://www.bioperl.org) and Ensembl Perl modules on a Linux platform. All of the phylogenetic trees and alignments can be found in the Supplemental materials.

Acknowledgments

We thank Christopher W.J. Smith and Juan Valcárcel for critical discussions, and Carol Featherston for editing of the manuscript. We are also grateful to Ângela Relógio and Christine Gemünd (EMBL) for the splicing factors' database and Claudia Ben-Dov, Carlos Caldas, Alan Christoffels, João Ferreira, Filipa Gallo, Margarida Gama-Carvalho, Inês Mollet, Teresa R. Pacheco, Søren Steffensen, Natalie Thorne, and Damian Yap for valuable contributions to this work. This work was supported by the Wellcome Trust, UK and Fundação para a Ciência e a Tecnologia, Portugal (Fellowship SFRH/BD/2914/2000).

Article published online ahead of print. Article and publication date are at http://www.genome.org/cgi/doi/10.1101/gr.3936206.

Footnotes

[Supplemental material is available online at www.genome.org.]

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Web site references

  1. http://www.ensembl.org; Ensembl.
  2. http://www.ebi.ac.uk/Wise2; Wise2—Intelligent algorithms for DNA searches (EBI).
  3. http://woody.embl-heidelberg.de/gene2est; Gene2EST BLAST Server.
  4. http://www.ncbi.nlm.nih.gov/BLAST; NCBI BLAST.
  5. http://www.es.embnet.org/Doc/SNAP; SNAP.pl (Synonymous Nonsynonymous Analysis Program).
  6. http://www.repeatmasker.org; RepeatMasker.
  7. http://www.sanger.ac.uk/Software/Pfam; Pfam—Protein families database of alignments and HMMs.
  8. http://smart.embl-heidelberg.de; SMART—Simple Modular Architecture Research Tool. [DOI] [PMC free article] [PubMed]
  9. http://www.bioperl.org; BioPerl.

Articles from Genome Research are provided here courtesy of Cold Spring Harbor Laboratory Press

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