Biology subjects

Speroni, S.

Publications and source records attributed to Speroni, S..

3 recordsLinked to original sources

bioRxiv2024

NuMA is a mitotic adaptor protein that activates dynein and connects it to microtubule minus ends

Nuclear mitotic apparatus protein (NuMA) is indispensable for the mitotic functions of the major microtubule minus-end directed motor cytoplasmic dynein 1. NuMA and dynein are both essential for correct spindle pole organization. How these proteins cooperate to gather microtubule minus ends at spindle poles remains unclear. Here we use microscopy-based in vitro reconstitutions to demonstrate that NuMA is a dynein adaptor, activating processive dynein motility together with dyneins cofactors dynactin and Lissencephaly-1 (Lis1). Additionally, we find that NuMA binds and stabilizes microtubule minus ends, allowing dynein/dynactin/NuMA to transport microtubule minus ends as cargo to other minus ends. We further show that the microtubule-nucleating {gamma}-tubulin ring complex ({gamma}TuRC) hinders NuMA binding. This shows that either {gamma}TuRC needs to be released or microtubules need to be severed to generate free minus ends before they can be incorporated into spindle poles by dynein/dynactin/NuMA. These results provide new mechanistic insight into how dynein, dynactin, NuMA and Lis1 cooperate to organize spindle poles in cells.

biochemistry↗

bioRxiv2022

APC couples neuronal mRNAs to multiple kinesins and EB1 for bidirectional mRNA movements along microtubules

Understanding where in the cytoplasm mRNAs are translated is increasingly recognised as being as important as knowing the timing and level of protein expression. mRNAs are localised via active motor-driven transport along microtubules (MTs) but the underlying essential factors and dynamic interactions are largely unknown. Using biochemical in vitro reconstitutions with purified mammalian proteins, multi-colour TIRF-microscopy (TIRF-M), and interaction kinetics measurements, we show that adenomatous polyposis coli (APC) enables kinesin-1- and kinesin-2-based mRNA transport, and that APC is an ideal adaptor for long-range mRNA transport as it forms highly stable complexes with 3UTR fragments of several neuronal mRNAs (APC-RNPs). The kinesin-1 KIF5A binds and transports several neuronal mRNP components such as FMRP, PUR, and mRNA fragments weakly, whereas the transport frequency of the mRNA fragments is significantly increased by APC. APC-RNP-motor complexes can assemble on MTs, generating highly processive mRNA transport events. We further find that EB1 recruits APC-RNPs to dynamically growing MT ends and APC-RNPs track shrinking MTs, producing MT minus-end-directed RNA motility due to the high dwell times of APC on MTs. Our findings establish APC as a versatile mRNA-kinesin adaptor and a key factor for the assembly and bidirectional movement of neuronal transport mRNPs.

biochemistry↗

bioRxiv2020

rec-Y2H matrix screening reveals a vast potential for direct protein-protein interactions among RNA binding proteins

RNA-binding proteins (RBPs) are crucial factors of post-transcriptional gene regulation and their modes of action are intensely investigated. At the center of attention are RNA motifs that guide where RBPs bind. However, sequence motifs are often poor predictors of RBP-RNA interactions in vivo. It is hence believed that many RBPs recognize RNAs as complexes, to increase specificity and regulatory possibilities. To probe the potential for complex formation among RBPs, we assembled a library of 978 mammalian RBPs and used rec-Y2H screening to detect direct interactions between RBPs, sampling > 600 K interactions. We discovered 1994 new interactions and demonstrate that interacting RBPs bind RNAs adjacently in vivo. We further find that the mRNA binding region and motif preferences of RBPs can deviate, depending on their adjacently binding interaction partners. Finally, we reveal novel RBP interaction networks among major RNA processing steps and show that splicing impairing RBP mutations observed in cancer rewire spliceosomal interaction networks. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=132 SRC="FIGDIR/small/296160v2_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@7a2dc6org.highwire.dtl.DTLVardef@1533c0dorg.highwire.dtl.DTLVardef@18bb4a1org.highwire.dtl.DTLVardef@ffe601_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗