Biology subjects

Somma, M. P.

Publications and source records attributed to Somma, M. P..

3 recordsLinked to original sources

bioRxiv2025

Distinct Cellular Effects of Myotonic Dystrophy type 2 RAN Tetrapeptides in Drosophila melanogaster

Myotonic dystrophy type 2 (DM2) is an autosomal dominant, multisystemic disorder caused by the expansion of CCTG repeats in the first intron of the CNBP gene. Repeat-associated non-AUG (RAN) translation of the expanded CCTG RNA may produce two tetrapeptide repeat proteins (TPRs), poly-QAGR and poly-PACL, whose roles in DM2 pathogenesis remain poorly understood. To investigate their individual contributions, we expressed codon-optimized QAGR and PACL peptides with ATG start codon in Drosophila melanogaster. Expression of QAGR and PACL TPRs significantly compromised fly viability and lifespan, induces eye degeneration and locomotor defects. We found that QAGR accumulated in the nucleolus, disrupted nucleolar integrity, and compromised rRNA processing. Moreover, QAGR expression interfered with autophagy, leading to the accumulation of Atg8a- and Ref(2)P-positive aggregates. Genetic interaction studies showed that overexpression of Atg8a or Ref(2)P mitigated QAGR-induced eye-toxicity, while knockdown of autophagy genes exacerbated it. Conversely, PACL repeats promoted stress granule formation, as indicated by their colocalization with TIAR in human cells and their epistatic interaction with the Drosophila orthologue Rox8. Notably, cytoplasmic PACL aggregates were observed in myoblasts of DM2 patient. Together these findings demonstrate that QAGR and PACL peptides exert distinct toxic effects, impairing nucleolar function and autophagy, or altering stress granule dynamics, respectively. Both mechanisms likely converge to drive DM2 pathogenesis and represent promising therapeutic targets.

genetics↗

bioRxiv2025

Microcephaly-Associated Genes asp and Sas4 Control Chromatin Organization and Nuclear Lamina Structure in Drosophila melanogaster

Autosomal recessive primary microcephaly (MCPH) is a neurodevelopmental disorder characterized by reduced brain size and non-progressive intellectual disability. Mutations in over 30 genes have been linked to MCPH. Nearly a half of the genes identified by these mutations encode proteins involved in centrosome biogenesis or microtubule (MT) dynamics, suggesting a central role for mitotic spindle organization and division plane orientation in disease aetiology. However, it has been suggested that disruptions in spindle positioning alone are not sufficient to lead to microcephaly. Here, we investigate the contribution of the Drosophila orthologs of ASPM/MCPH5 (asp) and CENPJ/MCPH6 (Sas4) to nuclear architecture, chromatin organization, and genome stability. We show that loss of either Sas4 or Asp leads to aberrant microtubule architecture, mislocalization of the LINC complex, and deformation of the nuclear lamina. These defects are accompanied by reduced levels of both lamin and HP1 and impaired centromere clustering in interphase cells. Sas4 and asp mutants also exhibit a global reduction in heterochromatin-associated histone marks (H3K9me2/3 and H3K27me3) and increased levels of the euchromatin-associated mark H3K4me3. Remarkably, treatment with Methylstat, a demethylase inhibitor, reduced nuclear invaginations by partially restoring H3K9me3 levels. Additionally, Sas4 or Asp depletion leads to DNA damage, increased sensitivity to genotoxic stress, and delayed DNA repair. Together, these findings reveal a previously underappreciated role for Asp and Sas4 in preserving nuclear architecture and chromatin integrity, offering new insight into the pathogenesis of MCPH. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=140 SRC="FIGDIR/small/666102v1_ufig1.gif" ALT="Figure 1"> View larger version (79K): org.highwire.dtl.DTLVardef@bfdfbborg.highwire.dtl.DTLVardef@f983eaorg.highwire.dtl.DTLVardef@143857borg.highwire.dtl.DTLVardef@1f0895b_HPS_FORMAT_FIGEXP M_FIG C_FIG

genetics↗

bioRxiv2021

Genetic control of kinetochore-driven microtubule growth in Drosophila mitosis

Centrosome-containing cells assemble their spindles exploiting three main classes of microtubules (MTs): MTs nucleated by the centrosomes, MTs generated near the chromosomes/kinetochores, and MTs nucleated within the spindle by the augmin-dependent pathway. Mammalian and Drosophila cells lacking the centrosomes generate MTs at kinetochores and eventually form functional bipolar spindles. However, the mechanisms underlying kinetochore-driven MT formation are poorly understood. One of the ways to elucidate these mechanisms is the analysis of spindle reassembly following MT depolymerization. Here, we used an RNA interference (RNAi)-based reverse genetics approach to dissect the process of kinetochore-driven MT regrowth (KDMTR) after colcemid-induced MT depolymerization. This MT depolymerization procedure allows a clear assessment of KDMTR, as colcemid disrupts centrosome-driven MT regrowth but allows KDMTR. We examined KDMTR in normal Drosophila S2 cells and in S2 cells subjected to RNAi against conserved genes involved in mitotic spindle assembly: mast/orbit/chb (CLASP1), mei-38 (TPX2), mars (HURP), dgt6 (HAUS6), Eb1 (MAPRE1/EB1), Patronin (CAMSAP2), asp (ASPM) and Klp10A (KIF2A). RNAi-mediated depletion of Mast/Orbit, Mei-38, Mars, Dgt6 and Eb1 caused a significant delay in KDMTR, while loss of Patronin had a milder negative effect on this process. In contrast, Asp or Klp10A deficiency increased the rate of KDMTR. These results coupled with the analysis of GFP-tagged proteins (Mast/Orbit, Mei-38, Mars, Eb1, Patronin and Asp) localization during KDMTR suggested a model for kinetochore-dependent spindle reassembly. We propose that kinetochores capture the plus ends of MTs nucleated in their vicinity and that these MTs elongate at kinetochores through the action of Mast/Orbit. The Asp protein binds the MT minus ends since the beginning of KDMTR, preventing excessive and disorganized MT regrowth. Mei-38, Mars, Dgt6, Eb1 and Patronin positively regulate polymerization, bundling and stabilization of regrowing MTs until a bipolar spindle is reformed. Author summaryThe mitotic spindle is a microtubule (MT)-based molecular machine that mediates precise chromosome segregation during cell division. Both Drosophila and human cells assemble their spindles exploiting two main classes of MTs: MTs nucleated by the centrosomes (MT nucleating organelles) and MTs generated at or near the kinetochores (the chromosome-associated structures that bind the spindle MTs). Cells of both species can assemble a functional mitotic spindle in the complete absence of centrosomes, but the mechanisms underlying this process are still poorly understood. We used Drosophila S2 cells as model system to analyze spindle reassembly following colcemid-induced MT depolymerization. MT regrowth (MTR) after colcemid treatment was particularly informative as this drug disrupts the MT nucleating ability of the centrosomes but allows kinetochore-driven MTR (KDMTR). We analyzed KDMTR in normal cells and in cells subjected to RNA interference (RNAi)-mediated depletion of 8 different evolutionarily conserved proteins involved in spindle assembly, and identified proteins that either promote or delay KDMTR. These results coupled with the analysis of proteins localization during spindle reassembly allowed us to integrate the current model on the role of kinetochore-driven MT growth in spindle formation.

genetics↗