Linear motifs: lost in (pre)translation

Pretranslational modification by alternative splicing, alternative promoter usage and RNA editing enables the production of multiple protein isoforms from a single gene. A large quantity of data now supports the notion that short linear motifs (SLiMs), which are protein interaction modules enriched within intrinsically disordered regions, are key for the functional diversification of these isoforms. The inclusion or removal of these SLiMs can switch the subcellular localisation of an isoform, promote cooperative associations, refine the affinity of an interaction, coordinate phase transitions within the cell, and even create isoforms of opposing function. This article discusses the novel functionality enabled by the addition or removal of SLiM-containing exons by pretranslational modifications, such as alternative splicing and alternative promoter usage, and how these alterations enable the creation and modulation of complex regulatory and signalling pathways.     

Tissue-specific splicing of disordered segments that embed binding motifs rewires protein interaction networks

Alternative inclusion of exons increases the functional diversity of proteins. Among alternatively spliced exons, tissue-specific exons play a critical role in maintaining tissue identity. This raises the question of how tissue-specific protein-coding exons influence protein function. Here we investigate the structural, functional, interaction, and evolutionary properties of constitutive, tissue-specific, and other alternative exons in human. We find that tissue-specific protein segments often contain disordered regions, are enriched in posttranslational modification sites, and frequently embed conserved binding motifs. Furthermore, genes containing tissue-specific exons tend to occupy central positions in interaction networks and display distinct interaction partners in the respective tissues, and are enriched in signaling, development, and disease genes. Based on these findings, we propose that tissue-specific inclusion of disordered segments that contain binding motifs rewires interaction networks and signaling pathways. In this way, tissue-specific splicing may contribute to functional versatility of proteins and increases the diversity of interaction networks across tissues.     

Multiple Modes of Protein–Protein Interactions Promote RNP Granule Assembly

Eukaryotic cells are known to contain a wide variety of RNA–protein assemblies, collectively referred to as RNP granules. RNP granules form from a combination of RNA–RNA, protein–RNA, and protein–protein interactions. In addition, RNP granules are enriched in proteins with intrinsically disordered regions (IDRs), which are frequently appended to a well-folded domain of the same protein. This structural organization of RNP granule components allows for a diverse set of protein–protein interactions including traditional structured interactions between well-folded domains, interactions of short linear motifs in IDRs with the surface of well-folded domains, interactions of short motifs within IDRs that weakly interact with related motifs, and weak interactions involving at most transient ordering of IDRs and folded domains with other components. In addition, both well-folded domains and IDRs in granule components frequently interact with RNA and thereby can contribute to RNP granule assembly. We discuss the contribution of these interactions to liquid–liquid phase separation and the possible role of phase separation in the assembly of RNP granules. We expect that these principles also apply to other non-membrane bound organelles and large assemblies in the cell.     

Distinct Types of Disorder in the Human Proteome: Functional Implications for Alternative Splicing

Intrinsically disordered regions have been associated with various cellular processes and are implicated in several human diseases, but their exact roles remain unclear. We previously defined two classes of conserved disordered regions in budding yeast, referred to as “flexible” and “constrained” conserved disorder. In flexible disorder, the property of disorder has been positionally conserved during evolution, whereas in constrained disorder, both the amino acid sequence and the property of disorder have been conserved. Here, we show that flexible and constrained disorder are widespread in the human proteome, and are particularly common in proteins with regulatory functions. Both classes of disordered sequences are highly enriched in regions of proteins that undergo tissue-specific (TS) alternative splicing (AS), but not in regions of proteins that undergo general (i.e., not tissue-regulated) AS. Flexible disorder is more highly enriched in TS alternative exons, whereas constrained disorder is more highly enriched in exons that flank TS alternative exons. These latter regions are also significantly more enriched in potential phosphosites and other short linear motifs associated with cell signaling. We further show that cancer driver mutations are significantly enriched in regions of proteins associated with TS and general AS. Collectively, our results point to distinct roles for TS alternative exons and flanking exons in the dynamic regulation of protein interaction networks in response to signaling activity, and they further suggest that alternatively spliced regions of proteins are often functionally altered by mutations responsible for cancer.     

The Balancing Act of Intrinsically Disordered Proteins: Enabling Functional Diversity while Minimizing Promiscuity

Intrinsically disordered proteins (IDPs) or regions (IDRs) perform diverse cellular functions, but are also prone to forming promiscuous and potentially deleterious interactions. We investigate the extent to which the properties of, and content in, IDRs have adapted to enable functional diversity while limiting interference from promiscuous interactions in the crowded cellular environment. Information on protein sequences, their predicted intrinsic disorder, and 3D structure contents is related to data on protein cellular concentrations, gene co-expression, and protein–protein interactions in the well-studied yeast Saccharomyces cerevisiae. Results reveal that both the protein IDR content and the frequency of “sticky” amino acids in IDRs (those more frequently involved in protein interfaces) decrease with increasing protein cellular concentration. This implies that the IDR content and the amino acid composition of IDRs experience negative selection as the protein concentration increases. In the S. cerevisiae protein–protein interaction network, the higher a protein's IDR content, the more frequently it interacts with IDR-containing partners, and the more functionally diverse the partners are. Employing a clustering analysis of Gene Ontology terms, we newly identify ~ 600 putative multifunctional proteins in S. cerevisiae. Strikingly, these proteins are enriched in IDRs and contribute significantly to all the observed trends. In particular, IDRs of multi-functional proteins feature more sticky amino acids than IDRs of their non-multifunctional counterparts, or the surfaces of structured yeast proteins. This property likely affords sufficient binding affinity for the functional interactions, commonly mediated by short IDR segments, thereby counterbalancing the loss in overall IDR conformational entropy upon binding.     

A Conserved Deubiquitinating Enzyme Uses Intrinsically Disordered Regions to Scaffold Multiple Protein Interaction Sites

In the canonical view of protein function, it is generally accepted that the three-dimensional structure of a protein determines its function. However, the past decade has seen a dramatic growth in the identification of proteins with extensive intrinsically disordered regions (IDRs), which are conformationally plastic and do not appear to adopt single three-dimensional structures. One current paradigm for IDR function is that disorder enables IDRs to adopt multiple conformations, expanding the ability of a protein to interact with a wide variety of disparate proteins. The capacity for many interactions is an important feature of proteins that occupy the hubs of protein networks, in particular protein-modifying enzymes that usually have a broad spectrum of substrates. One such protein modification is ubiquitination, where ubiquitin is attached to proteins through ubiquitin ligases (E3s) and removed through deubiquitinating enzymes. Numerous proteomic studies have found that thousands of proteins are dynamically regulated by cycles of ubiquitination and deubiquitination. Thus, how these enzymes target their wide array of substrates is of considerable importance for understanding the function of the cell''s diverse ubiquitination networks. Here, we characterize a yeast deubiquitinating enzyme, Ubp10, that possesses IDRs flanking its catalytic protease domain. We show that Ubp10 possesses multiple, distinct binding modules within its IDRs that are necessary and sufficient for directing protein interactions important for Ubp10''s known roles in gene silencing and ribosome biogenesis. The human homolog of Ubp10, USP36, also has IDRs flanking its catalytic domain, and these IDRs similarly contain binding modules important for protein interactions. This work highlights the significant protein interaction scaffolding abilities of IDRs in the regulation of dynamic protein ubiquitination.     

Evidence for the Concerted Evolution between Short Linear Protein Motifs and Their Flanking Regions

Background

Linear motifs are short modules of protein sequences that play a crucial role in mediating and regulating many protein–protein interactions. The function of linear motifs strongly depends on the context, e.g. functional instances mainly occur inside flexible regions that are accessible for interaction. Sometimes linear motifs appear as isolated islands of conservation in multiple sequence alignments. However, they also occur in larger blocks of sequence conservation, suggesting an active role for the neighbouring amino acids.

Results

The evolution of regions flanking 116 functional linear motif instances was studied. The conservation of the amino acid sequence and order/disorder tendency of those regions was related to presence/absence of the instance. For the majority of the analysed instances, the pairs of sequences conserving the linear motif were also observed to maintain a similar local structural tendency and/or to have higher local sequence conservation when compared to pairs of sequences where one is missing the linear motif. Furthermore, those instances have a higher chance to co–evolve with the neighbouring residues in comparison to the distant ones. Those findings are supported by examples where the regulation of the linear motif–mediated interaction has been shown to depend on the modifications (e.g. phosphorylation) at neighbouring positions or is thought to benefit from the binding versatility of disordered regions.

Conclusion

The results suggest that flanking regions are relevant for linear motif–mediated interactions, both at the structural and sequence level. More interestingly, they indicate that the prediction of linear motif instances can be enriched with contextual information by performing a sequence analysis similar to the one presented here. This can facilitate the understanding of the role of these predicted instances in determining the protein function inside the broader context of the cellular network where they arise.     

Codon usage is less optimized in eukaryotic gene segments encoding intrinsically disordered regions than in those encoding structural domains

Codon usage tends to be optimized in highly expressed genes. A plausible explanation for this phenomenon is that translational accuracy is increased in highly expressed genes with infrequent use of rare codons. Besides structural domains (SDs), eukaryotic proteins generally have intrinsically disordered regions (IDRs) that by themselves do not assume unique three-dimensional structures. As IDRs are free from structural constraint, they can probably accommodate more translational errors than SDs can. Thus, codon usage in IDRs is likely to be less optimized than that in SDs. Codon usage in all the genes of seven eukaryotes was examined in terms of both tRNA adaptation index and codon adaptation index. Different amino acid compositions in different protein regions were taken into account in calculating expected adaptation indices, to which observed indices were compared. Codon usage is less optimized in gene regions encoding IDRs than in those corresponding to SDs. The finding does not depend on whether IDRs are located at the N-terminus, in the middle, or at the C-terminus of proteins. Furthermore, the observation remains unchanged in two different algorithms used to predict IDRs in proteins. The result is consistent with the idea that IDRs tolerate more translational errors than SDs.     

Discovering molecular features of intrinsically disordered regions by using evolution for contrastive learning

A major challenge to the characterization of intrinsically disordered regions (IDRs), which are widespread in the proteome, but relatively poorly understood, is the identification of molecular features that mediate functions of these regions, such as short motifs, amino acid repeats and physicochemical properties. Here, we introduce a proteome-scale feature discovery approach for IDRs. Our approach, which we call “reverse homology”, exploits the principle that important functional features are conserved over evolution. We use this as a contrastive learning signal for deep learning: given a set of homologous IDRs, the neural network has to correctly choose a held-out homolog from another set of IDRs sampled randomly from the proteome. We pair reverse homology with a simple architecture and standard interpretation techniques, and show that the network learns conserved features of IDRs that can be interpreted as motifs, repeats, or bulk features like charge or amino acid propensities. We also show that our model can be used to produce visualizations of what residues and regions are most important to IDR function, generating hypotheses for uncharacterized IDRs. Our results suggest that feature discovery using unsupervised neural networks is a promising avenue to gain systematic insight into poorly understood protein sequences.     

Independent effects of alternative splicing and structural constraint on the evolution of mammalian coding exons

Alternative splicing (AS) is known to significantly affect exon-level protein evolutionary rates in mammals. Particularly, alternatively spliced exons (ASEs) have a higher nonsynonymous-to-synonymous substitution rate (dN/dS) ratio than constitutively spliced exons (CSEs), possibly because the former are required only occasionally for normal biological functions. Meanwhile, intrinsically disordered regions (IDRs), the protein regions lacking fixed 3D structures, are also reported to have an increased evolutionary rate due to lack of structural constraint. Interestingly, IDRs tend to be located in alternative protein regions. Yet which of these two factors is the major determinant of the increased dN/dS in mammalian ASEs remains unclear. By comparing human-macaque and human-mouse one-to-one orthologous genes, we demonstrate that AS and protein structural disorder have independent effects on mammalian exon evolution. We performed analyses of covariance to demonstrate that the slopes of the (dN/dS-percentage of IDR) regression lines differ significantly between CSEs and ASEs. In other words, the dN/dS ratios of both ASEs and CSEs increase with the proportion of IDR (PIDR), whereas ASEs have higher dN/dS ratios than CSEs when they have similar PIDRs. Since ASEs and IDRs may less frequently overlap with protein domains (which also affect dN/dS), we also examined the correlations between dN/dS ratio and exon type/PIDR by controlling for the density of protein domain. We found that the effects of exon type and PIDR on dN/dS are both independent of domain density. Our results imply that nature can select for different biological features with regard to ASEs and IDRs, even though the two biological features tend to be localized in the same protein regions.     

Intrinsically disordered regions in autophagy proteins

Autophagy is an essential eukaryotic pathway required for cellular homeostasis. Numerous key autophagy effectors and regulators have been identified, but the mechanism by which they carry out their function in autophagy is not fully understood. Our rigorous bioinformatic analysis shows that the majority of key human autophagy proteins include intrinsically disordered regions (IDRs), which are sequences lacking stable secondary and tertiary structure; suggesting that IDRs play an important, yet hitherto uninvestigated, role in autophagy. Available crystal structures corroborate the absence of structure in some of these predicted IDRs. Regions of orthologs equivalent to the IDRs predicted in the human autophagy proteins are poorly conserved, indicating that these regions may have diverse functions in different homologs. We also show that IDRs predicted in human proteins contain several regions predicted to facilitate protein–protein interactions, and delineate the network of proteins that interact with each predicted IDR‐containing autophagy protein, suggesting that many of these interactions may involve IDRs. Lastly, we experimentally show that a BCL2 homology 3 domain (BH3D), within the key autophagy effector BECN1 is an IDR. This BH3D undergoes a dramatic conformational change from coil to α‐helix upon binding to BCL2s, with the C‐terminal half of this BH3D constituting a binding motif, which serves to anchor the interaction of the BH3D to BCL2s. The information presented here will help inform future in‐depth investigations of the biological role and mechanism of IDRs in autophagy proteins. Proteins 2014; 82:565–578. © 2013 Wiley Periodicals, Inc.