SBDS is a complex disease linked to mutation in a single gene. Recent advances in genomics and functional bioinformatics are providing a new avenue for studying this and similar phenomena, complementing traditional hypothesis-driven laboratory research. Our bioinformatics approach undertaken in this study provides a new insight as to how a single gene mutation may affect diverse molecular functions, such as RNA metabolism, translation initiation, and cytoskeletal dynamics leading to complex disease.
Phylogenetic conservation of gene proximity may manifest itself in the presence of sets of orthologous genes in gene loci from different genomes. However, even in prokaryotes, it often comes up as conservation of positional connections between and inside particular pathways, not exactly the same representatives of the pathways.12
Therefore, integration of genomic data may uncover trends in gene positional clustering and the underlying functional links between genes and pathways. Analysis of our data points to initiation of translation as the main functional connection for SBDS (), with particular TYW1 and SBDS functional coupling as characteristic of all the studied vertebrate genomes.
Wybutosine is a hypermodified guanosine with a tricyclic base found at the 3′-position adjacent to the anticodon of eukaryotic phenylalanine tRNA. The UUU phenylalanine codon is highly prone to frameshift in the 3′ (rightward) direction at pyrimidine 3′ contexts,19
and wybutosine supports reading frame maintenance by stabilizing codon–anticodon interactions during decoding on the ribosome.20
Wybutosine synthesis might proceed through sequential reactions in a multiple protein complex assembled with the precursor tRNA and may be linked to exosomal/proteosomal structure that includes the SBDS protein. Comparative analysis of archeal sbds
loci stresses the potential involvement of SBDS in the exosomal complex where functions of translation, RNA processing, and degradation are tightly coupled.22
The SBDS protein has a thioredoxin fold, which might be directly coupled to the function of wybutosine reductase. Not all stages of wybutosine biosynthesis are known. There is some ambiguity surrounding the source of the C2 atom in wybutosine, suggested to originate from an intermediate in lysine metabolism. Interestingly, SBDS was shown to interact physically (Reactome db) with methylmalonate semialdehyde dehydrogenase, the enzyme in the branched-chain amino acid degradation pathway. It would be too speculative to go further, but the possible involvement of the SBDS protein in biosynthesis of wybutosine merits experimental validation.
Considering an alternative link between wybutosine and SBDS, we may suggest involvement of SBDS in degradation of noncharged or not properly modified tRNA. The tRNA surveillance pathway has been shown to exist in yeast and requires the exosome for polyadenylation and degradation of hypomodified pre-tRNA(i)(Met).23
Analogously, tRNA(Phe) without a modified wybutosine residue may be subject to degradation, and therefore SBDS may play some role in this process. The N-terminus of the Yhr087wp yeast protein has a fold similar to that in SBDS. It also has 2.4e-06 and 100.00% homology with bacterial tRNA pseudouridine 13 synthase, that may support an involvement of Yhr087wp itself and SBDS protein in tRNA modification. Genes do not cluster in S. cerevisiae
orthologs of SBDS (Q07953, SDO1) and TYW1 (Q08960, Tyw1p). However, the tyw1
locus in S. cerevisiae
also contains a gene for tRNA pseudouridine synthase 1 (YPL212C) and a number of genes encoding proteins of ribosomal biogenesis.
Establishing a link between SBDS and tRNA modification provides an interesting aspect of crosstalk between cytoskeleton function and translation machinery that may explain a faulty chemotactic phenotype of SBDS malfunction.
In the P. falciparum
and C. intestinalis
genomes, SBDS genes are colocalized with functions associated with tRNA-regulated components of translational initiation complexes.24
GCN1 is a translational activator and regulator of Gcn2p kinase activity. It forms a complex with Gcn20p and is proposed to stimulate Gcn2p activation by an uncharged tRNA.24
The second gene colocalized with sbds
in the sea squid genome is a gene for tRNA/RNA cytosine C5 methylase, which is required for initiating tRNA(i) (Met) modification. Eukaryotic and archeal initiation factors 2 are heterotrimeric proteins where only the γ subunit of the aIF2αβ
heterodimer contacts tRNA.26
Intriguingly, the aIF2β
gene is colocalized with sbds
in a genome of Plasmodium
Indirect evidence implicates actin as a cofactor in eukaryotic protein synthesis. The principle function of EF-1α is to bind aminoacyl-tRNA to the ribosome. EF-1α also interacts with the cytoskeleton by binding and bundling actin filaments and microtubules, and can alter the assembly of F-actin, a filamentous scaffold on which nonmembrane-associated protein translation take place27
(). F-actin and aa-tRNA compete for EF-1α, and their binding is pH-dependent and mutually exclusive. Release of EF-1α from actin binding was suggested to cause a transient increase in local concentration of the factor to facilitate polypeptide elongation. This interrelationship may ensure that cell proliferation and steady-state protein synthesis is separated from cell migration caused in primitive eukaryotic ancestors by starvation or by an avoidance response to other stressors.
Hypothetical functions of SBDS protein, and their effect on chemotaxis. Clouds represent gene proximity in different groups of organisms, ie, vertebrates (yellow), Ciona intestinalis (blue), and Plasmodium falciparum (purple).
There is also a crosstalk between the two systems in establishing cell polarity during chemotaxis. It has been proposed that the EF-1α-F-actin complex is an important scaffold for anchoring of β-actin mRNA to sites of active actin polymerization.28
Translation only occurs when the RNA-protein complex reaches its destination at the periphery of the cell.29
Nothing is known about localization of exosome complexes to cellular lamella, but the processes of translation and degradation of RNA species are likely to be colocalized and to be coordinated by cell locomotion.
It can be suggested that binding of tRNAphe with or without wybutosine modification differentially affects EF-1α. For example, tRNAphe without wybutosine modification can bind to EF-1α with a higher affinity, and eventually lead to cell polarization/arrest of movement. In this case, the function of the SBDS protein would be in downregulation or destruction of EF-1α, thereby inhibiting tRNA(s). It was shown recently that SBDS protein is also required for release and recycling of the nucleolar shuttling factor, Tif6, from pre-60S ribosomes,30
and also for pre-rRNA modifications and final maturation of the ribosome.31
Both processes can be regulated and coordinated with ribosomal transport to the cell periphery where actin translation and assembly takes place.32
We suggest a multiple involvement of SBDS protein in initiation and stability of translation in vertebrates. The suggested roles for this protein in wybutosine/tRNA metabolism would complement its potential involvement in the ribosome assembly recently reported for yeast and would crosstalk tightly, with establishment of cell polarity and cell locomotion. Experimental validation of the relationship between SBDS, wybutosine synthesis, and/or degradation of tRNAphe seems plausible, and we hope that our suggestions will attract the attention of biologists in related fields.
It is still not clear what functional features of a gene pair (structural or functional specificity of the encoded proteins, topology of their interaction, presence of a direct protein–protein contact) correlate significantly with their colocalization. Validation of genomic clustering of genes encoding metabolic functions demonstrates 90% correlation for yeast and just slightly less for a human genome. A high correlation was also shown between gene colocalization and their temporal and spatial expression profiles.14
All the existing studies point to phenotypic associations between genes clustered in genomes, but more information is required for a proper large-scale statistical analysis of the correlation. We hope that case-by-case analysis will support the general validity of this method and lead to a routine automatic approach to functional classification of eukaryotic proteins via systematic comparative genomics.