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Autoregulation of tubulin mRNA via reversible TTC5 sequestration by soluble ab-tubulins

ABSTRACT: Microtubule cytoskeleton, built from heterodimers of α and β-tubulins, is critically important to physically organize cells, mediate intracellular transport and power cell division. The availability of soluble αβ-tubulins influences the biomechanical properties and functions of microtubules. When present in excess, soluble αβ-tubulins induce degradation of their encoding mRNAs. This process involves the activation of a specificity factor TTC5, which recognizes nascent tubulins and recruits effectors that degrade the mRNA. But how TTC5 activity is regulated is unknown. Our biochemical and structural proteomic approaches reveal that soluble αβ-tubulins at steady-state levels sequester TTC5, repressing its activity. The carboxy-terminal domain of TTC5 acts as a molecular switch, toggling between soluble αβ-tubulin-bound and nascent tubulin-bound states. Loss of sequestration by soluble αβ-tubulins constitutively activates TTC5, leading to diminished tubulin mRNA levels and compromised microtubule-dependent chromosome segregation during cell division. Our findings provide a paradigm for how cells regulate the activity of a specificity factor to adapt posttranscriptional regulation of gene expression to cellular needs.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Homo Sapiens (human)

DISEASE(S): Neuroblastoma

SUBMITTER: Oscar Vadas

LAB HEAD: Oscar Vadas

PROVIDER: PXD050483 | Pride | 2024-10-18

REPOSITORIES: Pride

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			Action	DRS
	20220701_OT_HDXMS_06.mgf	Mgf
	20220701_OT_HDXMS_06.raw	Raw
	20220701_OT_HDXMS_08.raw	Raw
	20220701_OT_HDXMS_115.raw	Raw
	20220701_OT_HDXMS_117.raw	Raw

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Autoregulation of tubulin mRNA via reversible TTC5 sequestration by soluble αβ-tubulins

Project description:The microtubule cytoskeleton, built from heterodimers of α and β-tubulins, is critically important to physically organize cells, mediate intracellular transport and power cell division. The availability of soluble αβ-tubulins influences the biomechanical properties and dynamic functions of microtubules. When present in excess, soluble αβ-tubulins induce degradation of their encoding mRNAs. This process involves the specificity factor TTC5, which recognizes nascent tubulins and recruits effectors that degrade the mRNA. But how TTC5 activity is regulated is unknown. Our biochemical and structural proteomic approaches reveal that soluble αβ-tubulins sequester TTC5 at steady-state, repressing its activity. The carboxy-terminal domain of TTC5 acts as a molecular switch, toggling between soluble αβ-tubulin-bound and nascent tubulin-bound states. Loss-of-function mutantions that abolish sequestration by soluble αβ-tubulins constitutively activate TTC5, leading to diminished tubulin mRNA levels and compromised microtubule-dependent chromosome segregation during cell division. Our findings provide a paradigm for how cells regulate the activity of a specificity factor to adapt posttranscriptional regulation of gene expression to cellular needs.

2024-10-18 | PXD050557 | Pride

MS-based proteomic characterization of assembled microtubules

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A systematic screening pipeline identifies a new regulator of tyrosinated microtubule dynamics

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2022-08-16 | PXD030448 | Pride

Microtubules methylation LC-MSMS

Project description:Microtubules are critical for mitosis, cell motility, and protein and organelle transport, and are a validated target for anticancer drugs. However, tubulin regulation and recruitment in these cellular processes is less understood. Post-translational modifications of tubulin are proposed to regulate microtubule functions and dynamics. Although many such modifications have been investigated, tubulin methylations and enzymes responsible for methylation have only recently begun to be described. Here we report that N-lysine methyl transferase KMT5A (SET8/PR‑Set7), which methylates histone H4K20, also methylates α‑tubulin. Furthermore, the transcription factor LSF binds both tubulin and SET8, and enhances α-tubulin methylation in vitro, countered by FQI1, a specific small molecule inhibitor of LSF. Thus, the three SET8, LSF, and tubulin, all essential for mitotic progression, interact with each other. Overall, these results point to dual functions for both SET8 and LSF not only in chromatin regulation, but also for cytoskeletal modification.

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Mechanism of chaperone coordination during cotranslational protein folding in bacteria

Project description:Protein folding is assisted by molecular chaperones that bind nascent polypeptides during mRNA translation. Several structurally-distinct classes of chaperones promote de novo folding, suggesting that their activity is coordinated at the ribosome. Here we use biochemical reconstitution and structural proteomics to explore the molecular basis for cotranslational chaperone action in bacteria. We find that chaperone binding is disfavoured close to the ribosome, allowing folding to precede chaperone recruitment. Trigger factor subsequently recognises compact folding intermediates exposing extensive non-native surface and dictates DnaJ access to nascent chains. DnaJ uses a large surface to bind structurally diverse intermediates, and recruits DnaK to solvent-accessible sites in a sequence non-specific manner. Neither Trigger factor, DnaJ nor DnaK destabilize cotranslational folding intermediates. Instead, the chaperones collaborate to create a protected space for protein maturation that extends well beyond the ribosome exit tunnel. Our findings show how the chaperone network selects and modulates cotranslational folding intermediates.

2024-07-03 | PXD048638 | Pride

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Project description:The phylum of Apicomplexa groups intracellular parasites that employ substrate-dependent gliding motility to invade host cells, egress from the infected cells and cross biological barriers. The glideosome associated connector (GAC) is a conserved protein essential to this process. GAC facilitates the association of actin filaments with surface transmembrane adhesins and the efficient transmission of the force generated by myosin translocation of actin to the cell surface substrate. Here, we present the crystal structure of Toxoplasma gondii GAC and reveal a unique, supercoiled armadillo repeat region that adopts a closed ring conformation. Characterisation of the membrane binding interface within the C-terminal PH domain as well as an N-terminal fragment necessary for association with F-actin suggest that GAC adopts multiple conformations. A multi-conformational model for assembly of GAC within the glideosome is proposed

2023-05-10 | PXD039335 | Pride

Cross-linking of the microtubule binding N-terminal domain of the companion of cellulose synthase 1 protein (CC1∆C223) from Arabidopsis thaliana with tubulin

Project description:In this study, we use Cross-linking Mass Spectrometry to identify the interaction sites of the microtubule binding N-terminal domain of the companion of cellulose synthase 1 protein (CC1∆C223) from Arabidopsis thaliana with tubulin. We consistently detected four cross-linked peptides of CC1∆C223 to β-tubulin (K40 to E111, K94 to E111, K96 to E111 and K96 to E158; letters and numbers indicate specific amino acids in the protein sequence of CC1∆C223 and β-tubulin, respectively) and one cross-linked peptide of CC1∆C223 to α-tubulin (K40 to D327).

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An inhibitory segment within G-patch activators tunes Prp43-ATPase activity during ribosome assembly

Project description:Mechanisms by which G-patch activators tune the processive multi-tasking ATP-dependent RNA helicase Prp43 to remodel diverse RNA:protein complexes remain elusive. Here, a comparative study between a new activator, Tma23, and a characterized activator, Pxr1, uncovered segments that organize Prp43 function during ribosome assembly. In addition to the activating G-patch, we discovered an inhibitory segment within Tma23 and Pxr1, I-patch, that restrains Prp43-ATPase activity. Cryo-electron microscopy and hydrogen-deuterium exchange mass spectrometry show how I-patch binds to the catalytic RecA-like domains to allosterically inhibit Prp43-ATPase activity. Tma23 and Pxr1 also contain a dimerization segment which organizes Prp43 into higher-order complexes. We posit that Prp43 function at discrete locations on pre-ribosomal RNA are coordinated through toggling interactions with G-patch and I-patch motifs. This could guarantee measured and timely Prp43 activation, enabling precise control over multiple RNA remodeling events occurring concurrently during ribosome formation.

2024-10-09 | PXD046892 | Pride

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Differential modifications of the C-terminal tails of alpha-tubulin isoforms and their importance for kinesin-based microtubule transport in vivo

Project description:Microtubules (MTs) are built from alpha/beta-tubulin dimers and used as tracks by kinesin and dynein motors to transport a variety of cargos, such as mRNAs, proteins, and organelles, within the cell. Tubulins are subjected to several post-translational modifications (PTMs). Glutamylation is one of them, and it is responsible for adding one or more glutamic acid residues as branched peptide chains to the C-terminal tails of both alpha- and beta-tubulin. However, very little is known about the specific modifications found on the different tubulin isoforms in vivo and the role of these PTMs in cargo transport along MTs in vivo. In this study, we found that in Drosophila, glutamylation of the alpha-tubulin isoforms occurs specifically on the C-terminal ends of TBA1 and TBA3 in the ovaries. In contrast, the ovarian isoform TBA4 is not glutamylated. The C-terminal ends of TBA1 and TBA3 are glutamylated at several glutamyl side chains in various combinations. Drosophila TTLL5 is required for the mono- and polyglutamylation of ovarian TBA1 and 3. Furthermore, glutamylation of the alpha-tubulin is essential for the efficient localization of Staufen/osk mRNA and to give directionality to the fast ooplasmic streaming, two processes known to depend on kinesin mediated processes during oogenesis. In the nervous system, the kinesin-dependent neuronal transport of mitochondria also depends on TTLL5. Additionally, alpha-tubulin glutamylation affects the pausing of the transport of individual mitochondria in the axons. Our results demonstrate the in vivo role of TTLL5 in differential glutamylation of alpha-tubulin isoforms and point to the in vivo importance of alpha-tubulin glutamylation for kinesin-dependent processes.

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