Microtubules (MTs) undergo diverse posttranslational modifications that regulate their structural and functional properties. Among these, polyglutamylation-a dominant and conserved modification targeting unstructured tubulin C-terminal tails-plays a pivotal role in defining the tubulin code. Here, we describe a mechanism by which tubulin tyrosine ligase-like 11 (TTLL11) expands and diversifies the code. Cryo-electron microscopy revealed a unique bipartite MT recognition strategy wherein TTLL11 binding and catalytic domains engage adjacent MT protofilaments. Biochemical and cellular assays identified previously uncharacterized polyglutamylation patterns, showing that TTLL11 directly extends the primary polypeptide chains of α- and β-tubulin in vitro, challenging the prevailing paradigms emphasizing lateral branching. Moreover, cell-based and in vivo data suggest a cross-talk between polyglutamylation and the detyrosination/tyrosination cycle likely linked to the TTLL11-mediated elongation of the primary α-tubulin chain. These findings unveil an unrecognized layer of complexity within the tubulin code and offer mechanistic insights into the molecular basis of functional specialization of MT cytoskeleton.

• MeSH
• Cryoelectron Microscopy MeSH
• Humans MeSH
• Microtubules metabolism   MeSH
• Models, Molecular MeSH
• Peptide Synthases * metabolism   chemistry   genetics   MeSH
• Protein Processing, Post-Translational MeSH
• Tubulin * metabolism   chemistry   MeSH
• Protein Binding MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Peptide Synthases * MeSH
• tubulin polyglutamylase MeSH Browser
• Tubulin * MeSH

Retinitis pigmentosa (RP) is a hereditary disorder caused by mutations in more than 70 different genes including those that encode proteins important for pre-mRNA splicing. Most RP-associated mutations in splicing factors reduce either their expression, stability or incorporation into functional splicing complexes. However, we have previously shown that two RP mutations in PRPF8 (F2314L and Y2334N) and two in SNRNP200 (S1087L and R1090L) behaved differently, and it was still unclear how these mutations affect the functions of both proteins. To investigate this in the context of functional spliceosomes, we used iCLIP in HeLa and retinal pigment epithelial (RPE) cells. We found that both mutations in the RNA helicase SNRNP200 change its interaction with U4 and U6 snRNAs. The significantly broader binding profile of mutated SNRNP200 within the U4 region upstream of the U4/U6 stem I strongly suggests that its activity to unwind snRNAs is impaired. This was confirmed by FRAP measurements and helicase activity assays comparing mutant and WT protein. The RP variants of PRPF8 did not affect snRNAs, but showed a reduced binding to pre-mRNAs, which resulted in the slower splicing of introns and altered expression of hundreds of genes in RPE cells. This suggests that changes in the expression and splicing of specific genes are the main driver of retinal degeneration in PRPF8-linked RP.

• Keywords
• PRPF8, Pre-mRNA splicing, Retinitis pigmentosa, SNRNP200, iCLIP,
• MeSH
• HeLa Cells MeSH
• Humans MeSH
• Ribonucleoprotein, U4-U6 Small Nuclear metabolism   genetics   MeSH
• Mutation * MeSH
• RNA Precursors * metabolism   genetics   MeSH
• RNA-Binding Proteins * metabolism   genetics   MeSH
• Retinal Pigment Epithelium metabolism   MeSH
• Retinitis Pigmentosa * genetics   metabolism   pathology   MeSH
• Ribonucleoproteins, Small Nuclear * metabolism   genetics   MeSH
• RNA, Small Nuclear * metabolism   genetics   MeSH
• RNA Splicing genetics   MeSH
• RNA Splicing Factors metabolism   genetics   MeSH
• Protein Binding MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Ribonucleoprotein, U4-U6 Small Nuclear MeSH
• RNA Precursors * MeSH
• RNA-Binding Proteins * MeSH
• PRPF8 protein, human MeSH Browser
• Ribonucleoproteins, Small Nuclear * MeSH
• RNA, Small Nuclear * MeSH
• RNA Splicing Factors MeSH
• SNRNP200 protein, human MeSH Browser

BACKGROUND: Glutamate carboxypeptidase 2 (GCP2) belongs to the M28B metalloprotease subfamily encompassing a variety of zinc-dependent exopeptidases that can be found in many eukaryotes, including unicellular organisms. Limited information exists on the physiological functions of GCP2 orthologs in mammalian tissues outside of the brain and intestine, and such data are completely absent for non-mammalian species. Here, we investigate GCP2 orthologs found in trematodes, not only as putative instrumental molecules for defining their basal function(s) but also as drug targets. METHODS: Identified genes encoding M28B proteases Schistosoma mansoni and Fasciola hepatica genomes were analyzed and annotated. Homology modeling was used to create three-dimensional models of SmM28B and FhM28B proteins using published X-ray structures as the template. For S. mansoni, RT-qPCR was used to evaluate gene expression profiles, and, by RNAi, we exploited the possible impact of knockdown on the viability of worms. Enzymes from both parasite species were cloned for recombinant expression. Polyclonal antibodies raised against purified recombinant enzymes and RNA probes were used for localization studies in both parasite species. RESULTS: Single genes encoding M28B metalloproteases were identified in the genomes of S. mansoni and F. hepatica. Homology models revealed the conserved three-dimensional fold as well as the organization of the di-zinc active site. Putative peptidase activities of purified recombinant proteins were assayed using peptidic libraries, yet no specific substrate was identified, pointing towards the likely stringent substrate specificity of the enzymes. The orthologs were found to be localized in reproductive, digestive, nervous, and sensory organs as well as parenchymal cells. Knockdown of gene expression by RNAi silencing revealed that the genes studied were non-essential for trematode survival under laboratory conditions, reflecting similar findings for GCP2 KO mice. CONCLUSIONS: Our study offers the first insight to our knowledge into M28B protease orthologs found in trematodes. Conservation of their three-dimensional structure, as well as tissue expression pattern, suggests that trematode GCP2 orthologs may have functions similar to their mammalian counterparts and can thus serve as valuable models for future studies aimed at clarifying the physiological role(s) of GCP2 and related subfamily proteases.

• Keywords
• Fasciola hepatica, Folate hydrolase, Immunohistochemistry, M28B metalloproteases, NAALADase, Platyhelminth, Prostate specific-membrane antigen, RNA in situ hybridization, Schistosoma mansoni,
• MeSH
• Fasciola hepatica * genetics   MeSH
• Mice MeSH
• Peptide Hydrolases MeSH
• Mammals MeSH
• Schistosoma mansoni MeSH
• Trematoda * genetics   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• glutamate carboxypeptidase MeSH Browser
• Peptide Hydrolases MeSH