In silico methods for predicting functional synonymous variants.

Single nucleotide variants (SNVs) contribute to human genomic diversity. Synonymous SNVs are previously considered to be "silent," but mounting evidence has revealed that these variants can cause RNA and protein changes and are implicated in over 85 human diseases and cancers. Recent improvements in computational platforms have led to the development of numerous machine-learning tools, which can be used to advance synonymous SNV research. In this review, we discuss tools that should be used to investigate synonymous variants. We provide supportive examples from seminal studies that demonstrate how these tools have driven new discoveries of functional synonymous SNVs.

Implementing computational methods in tandem with synonymous gene recoding for therapeutic development.

Synonymous gene recoding, the substitution of synonymous variants into the genetic sequence, has been used to overcome many production limitations in therapeutic development. However, the safety and efficacy of recoded therapeutics can be difficult to evaluate because synonymous codon substitutions can result in subtle, yet impactful changes in protein features and require sensitive methods for detection. Given that computational approaches have made significant leaps in recent years, we propose that machine-learning (ML) tools may be leveraged to assess gene-recoded therapeutics and foresee an opportunity to adapt codon contexts to enhance some powerful existing tools. Here, we examine how synonymous gene recoding has been used to address challenges in therapeutic development, explain the biological mechanisms underlying its effects, and explore the application of computational platforms to improve the surveillance of functional variants in therapeutic design.

UBQLN proteins in health and disease with a focus on UBQLN2 in ALS/FTD.

Ubiquilin (UBQLN) proteins are a dynamic and versatile family of proteins found in all eukaryotes that function in the regulation of proteostasis. Besides their canonical function as shuttle factors in delivering misfolded proteins to the proteasome and autophagy systems for degradation, there is emerging evidence that UBQLN proteins play broader roles in proteostasis. New information suggests the proteins function as chaperones in protein folding, protecting proteins prior to membrane insertion, and as guardians for mitochondrial protein import. In this review, we describe the evidence for these different roles, highlighting how different domains of the proteins impart these functions. We also describe how changes in the structure and phase separation properties of UBQLNs may regulate their activity and function. Finally, we discuss the pathogenic mechanisms by which mutations in UBQLN2 cause amyotrophic lateral sclerosis and frontotemporal dementia. We describe the animal model systems made for different UBQLN2 mutations and how lessons learnt from these systems provide fundamental insight into the molecular mechanisms by which UBQLN2 mutations drive disease pathogenesis through disturbances in proteostasis.

ALS/FTD mutations in UBQLN2 are linked to mitochondrial dysfunction through loss-of-function in mitochondrial protein import.

UBQLN2 mutations cause amyotrophic lateral sclerosis (ALS) with frontotemporal dementia (FTD), but the pathogenic mechanisms by which they cause disease remain unclear. Proteomic profiling identified 'mitochondrial proteins' as comprising the largest category of protein changes in the spinal cord (SC) of the P497S UBQLN2 mouse model of ALS/FTD. Immunoblots confirmed P497S animals have global changes in proteins predictive of a severe decline in mitochondrial health, including oxidative phosphorylation (OXPHOS), mitochondrial protein import and network dynamics. Functional studies confirmed mitochondria purified from the SC of P497S animals have age-dependent decline in nearly all steps of OXPHOS. Mitochondria cristae deformities were evident in spinal motor neurons of aged P497S animals. Knockout (KO) of UBQLN2 in HeLa cells resulted in changes in mitochondrial proteins and OXPHOS activity similar to those seen in the SC. KO of UBQLN2 also compromised targeting and processing of the mitochondrial import factor, TIMM44, resulting in accumulation in abnormal foci. The functional OXPHOS deficits and TIMM44-targeting defects were rescued by reexpression of WT UBQLN2 but not by ALS/FTD mutant UBQLN2 proteins. In vitro binding assays revealed ALS/FTD mutant UBQLN2 proteins bind weaker with TIMM44 than WT UBQLN2 protein, suggesting that the loss of UBQLN2 binding may underlie the import and/or delivery defect of TIMM44 to mitochondria. Our studies indicate a potential key pathogenic disturbance in mitochondrial health caused by UBQLN2 mutations.

Single synonymous mutation in factor IX alters protein properties and underlies haemophilia B.

BACKGROUND: Haemophilia B is caused by genetic aberrations in the F9 gene. The majority of these are non-synonymous mutations that alter the primary structure of blood coagulation factor IX (FIX). However, a synonymous mutation c.459G>A (Val107Val) was clinically reported to result in mild haemophilia B (FIX coagulant activity 15%-20% of normal). The F9 mRNA of these patients showed no skipping or retention of introns and/or change in mRNA levels, suggesting that mRNA integrity does not contribute to the origin of the disease in affected individuals. The aim of this study is to elucidate the molecular mechanisms that can explain disease manifestations in patients with this synonymous mutation. METHODS: We analyse the molecular mechanisms underlying the FIX deficiency through in silico analysis and reproducing the c.459G>A (Val107Val) mutation in stable cell lines. Conformation and non-conformation sensitive antibodies, limited trypsin digestion, activity assays for FIX, interaction with other proteins and post-translation modifications were used to evaluate the biophysical and biochemical consequences of the synonymous mutation. RESULTS: The Val107Val synonymous mutation in F9 was found to significantly diminish FIX expression. Our results suggest that this mutation slows FIX translation and affects its conformation resulting in decreased extracellular protein level. The altered conformation did not change the specific activity of the mutated protein. CONCLUSIONS: The pathogenic basis for one synonymous mutation (Val107Val) in the F9 gene associated with haemophilia B was determined. A mechanistic understanding of this synonymous variant yields potential for guiding and developing future therapeutic treatments.

Multimerization of hepatitis delta antigen is a critical determinant of RNA binding specificity.

Hepatitis delta virus (HDV) RNA forms an unbranched rod structure that is associated with hepatitis delta antigen (HDAg) in cells replicating HDV. Previous in vitro binding experiments using bacterially expressed HDAg showed that the formation of a minimal ribonucleoprotein complex requires an HDV unbranched rod RNA of at least about 300 nucleotides (nt) and suggested that HDAg binds the RNA as a multimer of fixed size. The present study specifically examines the role of HDAg multimerization in the formation of the HDV ribonucleoprotein complex (RNP). Disruption of HDAg multimerization by site-directed mutagenesis was found to profoundly alter the nature of RNP formation. Mutant HDAg proteins defective for multimerization exhibited neither the 300-nt RNA size requirement for binding nor specificity for the unbranched rod structure. The results unambiguously demonstrate that HDAg binds HDV RNA as a multimer and that the HDAg multimer is formed prior to binding the RNA. RNP formation was found to be temperature dependent, which is consistent with conformational changes occurring on binding. Finally, analysis of RNPs constructed with unbranched rod RNAs successively longer than the minimum length indicated that multimeric binding is not limited to the first HDAg bound and that a minimum RNA length of between 604 and 714 nt is required for binding of a second multimer. The results confirm the previous proposal that HDAg binds as a large multimer and demonstrate that the multimer is a critical determinant of the structure of the HDV RNP.