Disorder-promoting Amino Acids Research Articles

Comparative Analysis of the Intrinsic Disorder Within the Layers of the Human Cornea.

The human cornea is essential for vision, providing structural integrity and refractive power to the eye. Recent advancements have deepened our understanding of the corneal molecular composition, yet the role of intrinsically disordered proteins within the cornea is unexplored. We analyzed 3,250 corneal proteins identified by Dyrlund et al, focusing on the epithelium, stroma, and endothelium layers. We performed a bioinformatics analysis to characterize the amino acid composition, the propensity for intrinsic protein disorder, and the distribution of protein types in 3 corneal layer proteome. Our study demonstrates that each corneal layer exhibited unique patterns in amino acid composition related to protein disorder. Order-promoting amino acids were generally depleted except for leucine, whereas disorder-promoting amino acids like arginine and glutamic acid were enriched across all layers. Significant variations were observed in the levels of intrinsic disorder among the different corneal layers, with substantial proportions of highly disordered proteins present in each. Analysis of protein class type in each layers revealed that no significant differences were detected in the distribution of protein classifications across the layers, suggesting a consistent population of the protein types across all corneal layers. Our findings reveal a sophisticated landscape of protein structures where intrinsic disorder varies across layers, suggesting an adaptation of the corneal proteome to the unique physiological demands of each layer. These structural variations may reflect the intricate requirements for corneal transparency, biomechanical stability, and environmental responsiveness.

IDPpred: a new sequence-based predictor for identification of intrinsically disordered protein with enhanced accuracy

Discovery of intrinsically disordered proteins (IDPs) and protein hybrids that contain both intrinsically disordered protein regions (IDPRs) along with ordered regions has changed the sequence–structure–function paradigm of protein. These proteins with lack of persistently fixed structure are often found in all organisms and play vital roles in various biological processes. Some of them are considered as potential drug targets due to their overrepresentation in pathophysiological processes. The major bottlenecks for characterizing such proteins are their occasional overexpression, difficulty in getting purified homogeneous form and the challenge of investigating them experimentally. Sequence-based prediction of intrinsic disorder remains a useful strategy especially for many large-scale proteomic investigations. However, worst accuracy still occurs for short disordered regions with less than ten residues, for the residues close to order–disorder boundaries, for regions that undergo coupled folding and binding in presence of partner, and for prediction of fully disordered proteins. Annotation of fully disordered proteins mostly relies on the far-UV circular dichroism experiment which gives overall secondary structure composition without residue-level resolution. Current methods including that using secondary structure information failed to predict half of target IDPs correctly in the recent Critical Assessment of protein Intrinsic Disorder prediction (CAID) experiment. This study utilized profiles of random sequential appearance of physicochemical properties of amino acids and random sequential appearance of order and disorder promoting amino acids in protein together with the existing CIDER feature for the prediction of IDP from sequence input. Our method was found to significantly outperform the existing predictors across different datasets.Communicated by Ramaswamy H. Sarma

The Cornified Envelope: A Versatile Contributor to the Epidermal Barrier

The Cornified Envelope: A Versatile Contributor to the Epidermal Barrier

Random peptides rich in small and disorder-promoting amino acids are less likely to be harmful.

Proteins are the workhorses of the cell, yet they carry great potential for harm via misfolding and aggregation. Despite the dangers, proteins are sometimes born de novo from noncoding DNA. Proteins are more likely to be born from noncoding regions that produce peptides that do little to no harm when translated than from regions that produce harmful peptides. To investigate which newborn proteins are most likely to “first, do no harm,” we estimate fitnesses from an experiment that competed Escherichia coli lineages that each expressed a unique random peptide. A variety of peptide metrics significantly predict lineage fitness, but this predictive power stems from simple amino acid frequencies rather than the ordering of amino acids. Amino acids that are smaller and that promote intrinsic structural disorder have more benign fitness effects. We validate that the amino acids that indicate benign effects in random peptides expressed in E. coli also do so in an independent data set of random N-terminal tags in which it is possible to control for expression level. The same amino acids are also enriched in young animal proteins.

A targeted bioinformatics approach identifies highly variable cell surface proteins that are unique to Glomeromycotina

Diversity in arbuscular mycorrhizal fungi (AMF) contributes to biodiversity and resilience in natural environments and healthy agricultural systems. Functional complementarity exists among species of AMF in symbiosis with their plant hosts, but the molecular basis of this is not known. We hypothesise this is in part due to the difficulties that current sequence assembly methodologies have assembling sequences for intrinsically disordered proteins (IDPs) due to their low sequence complexity. IDPs are potential candidates for functional complementarity because they often exist as extended (non-globular) proteins providing additional amino acids for molecular interactions. Rhizophagus irregularis arabinogalactan-protein-like proteins (AGLs) are small secreted IDPs with no known orthologues in AMF or other fungi. We developed a targeted bioinformatics approach to identify highly variable AGLs/IDPs in RNA-sequence datasets. The approach includes a modified multiple k-mer assembly approach (Oases) to identify candidate sequences, followed by targeted sequence capture and assembly (mirabait-mira). All AMF species analysed, including the ancestral family Paraglomeraceae, have small families of proteins rich in disorder promoting amino acids such as proline and glycine, or glycine and asparagine. Glycine- and asparagine-rich proteins also were found in Geosiphon pyriformis (an obligate symbiont of a cyanobacterium), from the same subphylum (Glomeromycotina) as AMF. The sequence diversity of AGLs likely translates to functional diversity, based on predicted physical properties of tandem repeats (elastic, amyloid, or interchangeable) and their broad pI ranges. We envisage that AGLs/IDPs could contribute to functional complementarity in AMF through processes such as self-recognition, retention of nutrients, soil stability, and water movement.

Antimicrobial Late Cornified Envelope Proteins: The Psoriasis Risk Factor Deletion of LCE3B/C Genes Affects Microbiota Composition

Late cornified envelope proteins are predominantly expressed in the skin and other cornified epithelia. On the basis of sequence similarity, this 18-member homologous gene family has been subdivided into six groups. The LCE3 proteins have been the focus of dermatological research because the combined deletion of LCE3B and LCE3C genes (LCE3B/C-del) is a risk factor for psoriasis. We previously reported that LCE3B/C-del increases the expression of the LCE3A gene and that LCE3 proteins exert antibacterial activity. In this study, we analyzed the antimicrobial properties of other family members and the role of LCE3B/C-del in the modulation of microbiota composition of the skin and oral cavity. Differences in killing efficiency and specificity between the late cornified envelope proteins and their target microbes were found, and the amino acid content rather than the order of the well-conserved central domain of the LCE3A protein was found responsible for its antibacterial activity. Invivo, LCE3B/C-del correlated with a higher beta-diversity in the skin and oral microbiota. From these results, we conclude that all late cornified envelope proteins possess antimicrobial activity. Tissue-specific and genotype-dependent antimicrobial protein profiles impact skin and oral microbiota composition, which could direct toward LCE3B/C-del‒associated dysbiosis and a possible role for microbiota in the pathophysiology of psoriasis.

Conservation and coevolution determine evolvability of different classes of disordered residues in human intrinsically disordered proteins.

Structure, function, and evolution are interdependent properties of proteins. Diversity of protein functions arising from structural variations is a potential driving force behind protein evolvability. Intrinsically disordered proteins or regions (IDPs or IDRs) lack well-defined structure under normal physiological conditions, yet, they are highly functional. Increased occurrence of IDPs in eukaryotes compared to prokaryotes indicates strong correlation of protein evolution and disorderedness. IDPs generally have higher evolution rate compared to globular proteins. Structural pliability allows IDPs to accommodate multiple mutations without affecting their functional potential. Nevertheless, how evolutionary signals vary between different classes of disordered residues (DRs) in IDPs is poorly understood. This study addresses variation of evolutionary behavior in terms of residue conservation and intra-protein coevolution among structural and functional classes of DRs in IDPs. Analyses are performed on 579 human IDPs, which are classified based on length of IDRs, interacting partners and functional classes. We find short IDRs are less conserved than long IDRs or full IDPs. Functional classes which require flexibility and specificity to perform their activity comparatively evolve slower than others. Disorder promoting amino acids evolve faster than order promoting amino acids. Pro, Gly, Ile, and Phe have unique coevolving nature which further emphasizes on their roles in IDPs. This study sheds light on evolutionary footprints in different classes of DRs from human IDPs and enhances our understanding of the structural and functional potential of IDPs.

Differential regulation of mRNA fate by the human Ccr4-Not complex is driven by coding sequence composition and mRNA localization

BackgroundRegulation of protein output at the level of translation allows for a rapid adaptation to dynamic changes to the cell’s requirements. This precise control of gene expression is achieved by complex and interlinked biochemical processes that modulate both the protein synthesis rate and stability of each individual mRNA. A major factor coordinating this regulation is the Ccr4-Not complex. Despite playing a role in most stages of the mRNA life cycle, no attempt has been made to take a global integrated view of how the Ccr4-Not complex affects gene expression.ResultsThis study has taken a comprehensive approach to investigate post-transcriptional regulation mediated by the Ccr4-Not complex assessing steady-state mRNA levels, ribosome position, mRNA stability, and protein production transcriptome-wide. Depletion of the scaffold protein CNOT1 results in a global upregulation of mRNA stability and the preferential stabilization of mRNAs enriched for G/C-ending codons. We also uncover that mRNAs targeted to the ER for their translation have reduced translational efficiency when CNOT1 is depleted, specifically downstream of the signal sequence cleavage site. In contrast, translationally upregulated mRNAs are normally localized in p-bodies, contain disorder-promoting amino acids, and encode nuclear localized proteins. Finally, we identify ribosome pause sites that are resolved or induced by the depletion of CNOT1.ConclusionsWe define the key mRNA features that determine how the human Ccr4-Not complex differentially regulates mRNA fate and protein synthesis through a mechanism linked to codon composition, amino acid usage, and mRNA localization.

Beyond Microsatellite Instability: Intrinsic Disorder as a Potential Link Between Protein Short Tandem Repeats and Cancer.

Short tandem repeats (STRs) are abundant in genomic sequences and are known for comparatively high mutation rates; STRs therefore are thought to be a potent source of genetic diversity. In protein-coding sequences STRs primarily encode disorder-promoting amino acids and are often located in intrinsically disordered regions (IDRs). STRs are frequently studied in the scope of microsatellite instability (MSI) in cancer, with little focus on the connection between protein STRs and IDRs. We believe, however, that this relationship should be explicitly included when ascertaining STR functionality in cancer. Here we explore this notion using all canonical human proteins from SwissProt, wherein we detected 3,699 STRs. Over 80% of these consisted completely of disorder promoting amino acids. 62.1% of amino acids in STR sequences were predicted to also be in an IDR, compared to 14.2% for non-repeat sequences. Over-representation analysis showed STR-containing proteins to be primarily located in the nucleus where they perform protein- and nucleotide-binding functions and regulate gene expression. They were also enriched in cancer-related signaling pathways. Furthermore, we found enrichments of STR-containing proteins among those correlated with patient survival for cancers derived from eight different anatomical sites. Intriguingly, several of these cancer types are not known to have a MSI-high (MSI-H) phenotype, suggesting that protein STRs play a role in cancer pathology in non MSI-H settings. Their intrinsic link with IDRs could therefore be an attractive topic of future research to further explore the role of STRs and IDRs in cancer. We speculate that our observations may be linked to the known dosage-sensitivity of disordered proteins, which could hint at a concentration-dependent gain-of-function mechanism in cancer for proteins containing STRs and IDRs.

Efficient secretory expression of Bacillus stearothermophilus α/β-cyclodextrin glycosyltransferase in Bacillus subtilis

Bacillus stearothermophilus α/β-cyclodextrin glycosyltransferase (α/β-CGTase) is an excellent transglycosylase with broad potential for food application, but its expression level is low in Bacillus subtilis. In this study, the optimal signal peptide for α/β-CGTase expression was screened from 173 signal peptides in B. subtilis WS11. The α/β-CGTase activity in a 3-L fermentor reached 151.93 U⋅ mL−1, but substantial amounts of inclusion bodies were produced. The N-terminal 12 amino acids of α/β-CGTase were then replaced with the N-terminal 15 amino acids of a β-CGTase from the same family that has a high percentage of disorder-promoting amino acids. As a result, the inclusion bodies were significantly reduced, and the enzyme activity increased to 249.35 U mL−1, 2.3 times that of the strain constructed previously. Finally, the ppsE and sfp genes of B. subtilis WS11, which are related to lipopeptide biosurfactant synthesis, were knocked out to produce B. subtilis WS13. When B. subtilis WS13 was used to produce α/β-CGTase in a 3-L fermentor, 70 % less defoaming agent was required than with B. subtilis WS11. Furthermore, enzyme production and growth of WS13 were equivalent to those of WS11. This study is of great significance for future research to efficiently scale-up production of α/β-CGTase.

A New Census of Protein Tandem Repeats and Their Relationship with Intrinsic Disorder

Protein tandem repeats (TRs) are often associated with immunity-related functions and diseases. Since that last census of protein TRs in 1999, the number of curated proteins increased more than seven-fold and new TR prediction methods were published. TRs appear to be enriched with intrinsic disorder and vice versa. The significance and the biological reasons for this association are unknown. Here, we characterize protein TRs across all kingdoms of life and their overlap with intrinsic disorder in unprecedented detail. Using state-of-the-art prediction methods, we estimate that 50.9% of proteins contain at least one TR, often located at the sequence flanks. Positive linear correlation between the proportion of TRs and the protein length was observed universally, with Eukaryotes in general having more TRs, but when the difference in length is taken into account the difference is quite small. TRs were enriched with disorder-promoting amino acids and were inside intrinsically disordered regions. Many such TRs were homorepeats. Our results support that TRs mostly originate by duplication and are involved in essential functions such as transcription processes, structural organization, electron transport and iron-binding. In viruses, TRs are found in proteins essential for virulence.

Mutually exclusive locales for N-linked glycans and disorder in human glycoproteins

Several post-translational protein modifications lie predominantly within regions of disorder: the biased localization has been proposed to expand the binding versatility of disordered regions. However, investigating a representative dataset of 500 human N-glycoproteins, we observed the sites of N-linked glycosylations or N-glycosites, to be predominantly present in the regions of predicted order. When compared with disordered stretches, ordered regions were not found to be enriched for asparagines, serines and threonines, residues that constitute the sequon signature for conjugation of N-glycans. We then investigated the basis of mutual exclusivity between disorder and N-glycosites on the basis of amino acid distribution: when compared with control ordered residue stretches without any N-glycosites, residue neighborhoods surrounding N-glycosites showed a depletion of bulky, hydrophobic and disorder-promoting amino acids and an enrichment for flexible and accessible residues that are frequently found in coiled structures. When compared with control disordered residue stretches without any N-glycosites, N-glycosite neighborhoods were depleted of charged, polar, hydrophobic and flexible residues and enriched for aromatic, accessible and order-promoting residues with a tendency to be part of coiled and β structures. N-glycosite neighborhoods also showed greater phylogenetic conservation among amniotes, compared with control ordered regions, which in turn were more conserved than disordered control regions. Our results lead us to propose that unique primary structural compositions and differential propensities for evolvability allowed for the mutual spatial exclusion of N-glycosite neighborhoods and disordered stretches.

Do sequence neighbours of intrinsically disordered regions promote structural flexibility in intrinsically disordered proteins?

Intrinsically disordered proteins (IDPs) are crucial players in various cellular activities. Several experimental and computational analyses have been conducted to study structural pliability and functional potential of IDPs. In spite of active research in past few decades, what induces structural disorder in IDPs and how is still elusive. Many studies testify that sequential and spatial neighbours often play important roles in determining structural and functional behaviour of proteins. Considering this fact, we assessed sequence neighbours of intrinsically disordered regions (IDRs) to understand if they have any role to play in inducing structural flexibility in IDPs. Our analysis includes 97% eukaryotic IDPs and 3% from bacteria and viruses. Physicochemical and structural parameters including amino acid propensity, hydrophobicity, secondary structure propensity, relative solvent accessibility, B-factor and atomic packing density are used to characterise the neighbouring residues of IDRs (NRIs). We show that NRIs exhibit a unique nature, which makes them stand out from both ordered and disordered residues. They show correlative occurrences of residue pairs like Ser-Thr and Gln-Asn, indicating their tendency to avoid strong biases of order or disorder promoting amino acids. We also find differential preferences of amino acids between N- and C-terminal neighbours, which might indicate a plausible directional effect on the dynamics of adjacent IDRs. We designed an efficient prediction tool using Random Forest to distinguish the NRIs from the ordered residues. Our findings will contribute to understand the behaviour of IDPs, and may provide potential lead in deciphering the role of IDRs in protein folding and assembly.

Identification and transcriptional analysis of dehydrin gene family in cucumber (Cucumis sativus)

Dehydrins (DHNs) are a group II late embryogenesis abundant (LEA) proteins that play essential roles in plant growth, development and responses to diverse environmental stimuli. Here, four DHNs in cucumber genome were identified using bioinformatics-based methods according to the highly conserved K-, Y- and S-segments, including 1 YnKn-type, 2 YnSKn-type, and 1 SKn-type DHNs. All of them are intrinsically disordered proteins (IDPs) and possess a large number of disorder-promoting amino acids. Secondary structure prediction revealed that each of them is composed of high proportion of alpha helix and random coil. Gene structure and phylogenetic analyses with DHNs from cucumber and several other species revealed that some closely related DHN genes had similar gene structures. A number of cis-elements involved in stress responses and phytohormones were found in each CsDHN promoter. The tissue expression profiles suggested that the CsDHN genes have overlapping, but different expression patterns. qRT-PCR results showed that three selected CsDHN genes could respond to heat, cold, osmotic and salt stresses, as well as to signaling molecules such as H2O2 and ABA. These results lay a solid foundation for future functional investigation of the cucumber dehydrin gene family in tissue development and stress responses in plants.

Effect of Neighbouring Residues in Conformational Plasticity of Intrinsically Disordered Proteins

Effect of neighbouring residues in conformational plasticity of intrinsically disordered regions. The concept of unstructured proteins has opened new avenues in the field of structural biology. Intrinsically disordered proteins (IDPs) are the new class of proteins which have been found to be a major player in many significant cellular functions. IDPs have been characterised by its physicochemical properties as well as its molecular interaction behaviour. Detailed study of IDPs can lead to a better understanding of protein folding and its functioning. To understand the source of disorderedness in the disordered regions (IDRs) in IDPs, we studied how the sequence environment of a disordered region correlates to its randomness. Here, we analysed the physicochemical and structural features like amino acid propensities, net charge, hydropathy index, secondary structure propensity, relative surface accessibility, interaction density and H-bonds to characterise the neighbours of the IDRs. Five residues, each towards N and C terminal of the disordered region are considered as the neighbours of IDRs. These neighbouring residues are found to be enriched in disorder promoting amino acids and have higher propensity to form loops than other secondary structures. Solvent accessibility of neighbouring residues also showed increasing trend as we move towards the IDRs. The variation of other parameters along with the above observation indicates that the neighbouring residues of IDRs induce a degree of flexibility to the adjoining IDRs. Based on our findings, we are designing an algorithm using random forest, which shall predict the disordered region based on its neighbouring sequences. The information on IDRs and its neighbours can be useful for proteins to be expressed or characterised for the first time. It can also provide a lead in understanding the molecular mechanism behind the polymorphic interactions that are involved with IDPs.

Molecular cloning and characterization of an ASR gene from Cucumis sativus

Abscisic acid-, stress- and ripening-induced (ASR) proteins are widely present in the plant kingdom and play important roles in different biological processes. However, no reports of ASR proteins are available in cucumber. In this study, an ASR gene (CsASR1) was identified and characterized from Cucumis sativus. CsASR1 exhibited a high content of disorder-promoting amino acids, indicating that it is an intrinsically disordered protein (IDP). CsASR1 protein was highly homologous to ASR proteins from other plant species. Expression of CsASR1 was induced by diverse abiotic stresses such as heat, PEG and NaCl, as well as by signaling molecules such as ABA and H2O2, suggesting a close relationship between CsASR1 and abiotic stress. Overexpression of CsASR1 could increase the tolerance against salinity and osmotic stress in E. coli. Transgenic Arabidopsis plants overexpressing CsASR1 exhibited higher germination rate than WT plants on MS medium containing various concentrations of NaCl. In addition, overexpression of CsASR1 in Arabidopsis resulted in significantly improved salt tolerance due to the increased activity of SOD and elevated transcripts of SOS3 and LEA4-5. Finally, CsASR1 could protect the activity of lactate dehydrogenase (LDH) from heat-induced inactivation. Taken together, our results demonstrate that CsASR1 plays an important role in abiotic stress tolerance, and it may function as an IDP to confer abiotic stress tolerance by protecting some stress-related proteins from inactivation under stress conditions.

The IDP-Specific Force Field ff14IDPSFF Improves the Conformer Sampling of Intrinsically Disordered Proteins.

Intrinsically disordered proteins (IDPs) or intrinsically disordered regions do not have a fixed tertiary structure but play key roles in signal regulation, molecule recognition, and drug targeting. However, it is difficult to study the structure and function of IDPs by traditional experimental methods because of their diverse conformations. Limitations of current generic protein force fields and solvent models were reported in the previous simulations of IDPs. We have also explored overcoming these limitations by developing the ff99IDPs and ff14IDPs force fields to correct the dihedral distribution for eight disorder-promoting residues often observed in IDPs and found encouraging improvements. Here we extend our correction of backbone dihedral terms to all 20 naturally occurring amino acids in the IDP-specific force field ff14IDPSFF to further improve the quality of the modeling of IDPs. Extensive tests of seven IDPs and 14 unstructured short peptides show that the simulated Cα chemical shifts obtained with the ff14IDPSFF force field are in quantitative agreement with those from NMR experiments and are more accurate than those obtained with the base generic force field and also our previous ff14IDPs that only corrects the eight disorder-promoting amino acids. The influence of the solvent model was also investigated and found to be less important. Finally, our explicit-solvent MD simulations further show that ff14IDPSFF can still be used to model structural and dynamical properties of two tested folded proteins, with a slightly better agreement in the loop regions for both structural and dynamical properties. These findings confirm that the newly developed IDP-specific force field ff14IDPSFF can improve the conformer sampling of intrinsically disordered proteins.

High GC content causes orphan proteins to be intrinsically disordered.

De novo creation of protein coding genes involves the formation of short ORFs from noncoding regions; some of these ORFs might then become fixed in the population. These orphan proteins need to, at the bare minimum, not cause serious harm to the organism, meaning that they should for instance not aggregate. Therefore, although the creation of short ORFs could be truly random, the fixation should be subjected to some selective pressure. The selective forces acting on orphan proteins have been elusive, and contradictory results have been reported. In Drosophila young proteins are more disordered than ancient ones, while the opposite trend is present in yeast. To the best of our knowledge no valid explanation for this difference has been proposed. To solve this riddle we studied structural properties and age of proteins in 187 eukaryotic organisms. We find that, with the exception of length, there are only small differences in the properties between proteins of different ages. However, when we take the GC content into account we noted that it could explain the opposite trends observed for orphans in yeast (low GC) and Drosophila (high GC). GC content is correlated with codons coding for disorder promoting amino acids. This leads us to propose that intrinsic disorder is not a strong determining factor for fixation of orphan proteins. Instead these proteins largely resemble random proteins given a particular GC level. During evolution the properties of a protein change faster than the GC level causing the relationship between disorder and GC to gradually weaken.

Is unphosphorylated Rex, as multifunctional protein of HTLV-1, a fully intrinsically disordered protein? An in silico study

Intracellularlocation of a viral unspliced mRNA in host cell is a crucial factor for normal life of the virus. Rex is a neucleo-cytoplasmic shuffling protein of Human T-cell Leukemia Virus-1(HTLV-1)which has important role in active transport of cargo-containing RNA from nucleus to cytoplasm. Therefore, it plays a crucial role in the disease development by the virus. In spite of its importance, the 3d-structurephosphorylated and unphosphorylated of this protein has not been determined. In this study, first we predicted whether Rex protein is an ordered or disordered protein. In second step protein 3Dstructure of Rex was obtained. The content of disorder-promoting amino acids, flexibility, hydrophobicity, short linear motifs (SLiMs) and protein binding regions and probability of Rex crystallization were calculated by various In Silico methods. The3D models of Rex protein were obtained by various In Silico methods, such as homology modeling, threading and ab initio, including; I-TASSER, LOMETS, SPARSKS, ROBBETA and QUARK servers. By comparing and analyzing Qmean, z-scores and energy levels of selected models, the best structures with highest favored region in Ramachandran plot (higher than 90%) was refined with MODREFINER software. In silico analysis of Rex physicochemical properties and also predicted SLiMs and binding regions sites confirms that unphosphorylated Rex protein in HTLV-1 as Rev protin in HIV is wholly disordered protein belongs to the class of intrinsically disordered proteins with extended disorder (native coils, native pre-molten globules).

MIR retroposon exonization promotes evolutionary variability and generates species-specific expression of IGF-1 splice variants

Insulin-like growth factor (IGF) -1 is a pleiotropic hormone exerting mitogenic and anti-apoptotic effects. Inclusion or exclusion of exon 5 into the IGF-1 mRNA gives rise to three transcripts, IGF-1Ea, IGF-1Eb and IGF-1Ec, which yield three different C-terminal extensions called Ea, Eb and Ec peptides. The biological significance of the IGF-1 splice variants and how the E-peptides affect the actions of mature IGF-1 are largely unknown. In this study we investigated the origin and conservation of the IGF-1 E-peptides and we compared the pattern of expression of the IGF-1 isoforms in vivo, in nine mammalian species, and in vitro using human and mouse IGF-1 minigenes.Our analysis showed that only IGF-1Ea is conserved among all vertebrates, whereas IGF-1Eb and IGF-1Ec are an evolutionary novelty originated from the exonization of a mammalian interspersed repetitive-b (MIR-b) element. Both IGF-1Eb and IGF-1Ec mRNAs were constitutively expressed in all mammalian species analyzed but their expression ratio varies greatly among species. Using IGF-1 minigenes we demonstrated that divergence in cis-acting regulatory elements between human and mouse conferred species-specific features to the exon 5 region. Finally, the protein-coding sequences of exon 5 showed low rate of synonymous mutations and contain disorder-promoting amino acids, suggesting a regulatory role for these domains.In conclusion, exonization of a MIR-b element in the IGF-1 gene determined gain of exon 5 during mammalian evolution. Alternative splicing of this novel exon added new regulatory elements at the mRNA and protein level potentially able to regulate the mature IGF-1 across tissues and species.