A nematode-specific ribonucleoprotein complex mediates interactions between the major nematode spliced leader snRNP and its target pre-mRNAs

Acknowledgements We thank Maheshika Kurukulasuriya for her assistance with immunoprecipitations from embryo extracts. Some strains were provided by the Caenorhabditis Genetics Center, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440). Sequencing was performed by the Centre for Genome-Enabled Biology and Medicine of the University of Aberdeen, and proteomics analysis by the Aberdeen proteomics facility. We thank Kate Burgoyne and Craig Pattinson (Aberdeen Proteomics) for technical support. We thank WormBase for providing the community resource that facilitated the interrogation of C. elegans molecular genetics used in this work . Author contribution: P.E., M.A., E.S.-M., R.F., M.W., B.M. and J.P. contributed experiments. J.P. and B.M. conceived the research and managed and coordinated the research activity; J.P. and B.M. acquired funding for the project; D.S. guided the proteomics analysis, M.W., B.M. and J.P. designed and implemented the computational analysis; M.W., B.M. and J.P. wrote the manuscript and prepared figures and tables.Peer reviewe

Development of BromoTag:A “Bump-&-Hole”-PROTAC system to induce potent, rapid, and selective degradation of tagged target proteins

[Image: see text] Small-molecule-induced protein depletion technologies, also called inducible degrons, allow degradation of genetically engineered target proteins within cells and animals. Here, we design and develop the BromoTag, a new inducible degron system comprising a Brd4 bromodomain L387A variant as a degron tag that allows direct recruitment by heterobifunctional bumped proteolysis targeting chimeras (PROTACs) to hijack the VHL E3 ligase. We describe extensive optimization and structure–activity relationships of our bump-and-hole–PROTACs using a CRISPR knock-in cell line expressing model target BromoTag-Brd2 at endogenous levels. Collectively, our cellular and mechanistic data qualifies bumped PROTAC AGB1 as a potent, fast, and selective degrader of BromoTagged proteins, with a favorable pharmacokinetic profile in mice. The BromoTag adds to the arsenal of chemical genetic degradation tools allowing us to manipulate protein levels to interrogate the biological function and therapeutic potential in cells and in vivo

A novel, essential trans-splicing protein connects the nematode SL1 snRNP to the CBC-ARS2 complex

Open Access via the OUP Agreement Some strains were provided by the Caenorhabditis Genetics Center, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440). Sequencing was performed by the Centre for Genome-Enabled Biology and Medicine of the University of Aberdeen, and proteomics analysis by the University of Aberdeen Proteomics facility. We thank WormBase for providing the community resource that facilitated the interrogation of C. elegans molecular genetics used in this work. FUNDING Biotechnology and Biological Sciences Research Council [BB/T002859/1]; EASTBIO Biotechnology and Biological Sciences Research Council PhD Studentship [BB/M010996/1 to R.E.B.S.]; University of Aberdeen Elphinstone PhD studentship (to R.Y.F.); University of Aberdeen. Funding for open access charge: Read and publish Agreement Scottish Institutions (SHEDL affiliated).Peer reviewedPublisher PD

Parkinson's VPS35[D620N] mutation induces LRRK2-mediated lysosomal association of RILPL1 and TMEM55B

We demonstrate that the Parkinson's VPS35[D620N] mutation alters the expression of ~220 lysosomal proteins and stimulates recruitment and phosphorylation of Rab proteins at the lysosome. This recruits the phospho-Rab effector protein RILPL1 to the lysosome where it binds to the lysosomal integral membrane protein TMEM55B. We identify highly conserved regions of RILPL1 and TMEM55B that interact and design mutations that block binding. In mouse fibroblasts, brain, and lung, we demonstrate that the VPS35[D620N] mutation reduces RILPL1 levels, in a manner reversed by LRRK2 inhibition and proteasome inhibitors. Knockout of RILPL1 enhances phosphorylation of Rab substrates, and knockout of TMEM55B increases RILPL1 levels. The lysosomotropic agent LLOMe also induced LRRK2 kinase-mediated association of RILPL1 to the lysosome, but to a lower extent than the D620N mutation. Our study uncovers a pathway through which dysfunctional lysosomes resulting from the VPS35[D620N] mutation recruit and activate LRRK2 on the lysosomal surface, driving assembly of the RILPL1-TMEM55B complex.</p

Parkinson's VPS35[D620N] mutation induces LRRK2-mediated lysosomal association of RILPL1 and TMEM55B

We demonstrate that the Parkinson's VPS35[D620N] mutation alters the expression of ~220 lysosomal proteins and stimulates recruitment and phosphorylation of Rab proteins at the lysosome. This recruits the phospho-Rab effector protein RILPL1 to the lysosome where it binds to the lysosomal integral membrane protein TMEM55B. We identify highly conserved regions of RILPL1 and TMEM55B that interact and design mutations that block binding. In mouse fibroblasts, brain, and lung, we demonstrate that the VPS35[D620N] mutation reduces RILPL1 levels, in a manner reversed by LRRK2 inhibition and proteasome inhibitors. Knockout of RILPL1 enhances phosphorylation of Rab substrates, and knockout of TMEM55B increases RILPL1 levels. The lysosomotropic agent LLOMe also induced LRRK2 kinase-mediated association of RILPL1 to the lysosome, but to a lower extent than the D620N mutation. Our study uncovers a pathway through which dysfunctional lysosomes resulting from the VPS35[D620N] mutation recruit and activate LRRK2 on the lysosomal surface, driving assembly of the RILPL1-TMEM55B complex.</p

An in vivo genetic screen for genes involved in spliced leader trans-splicing indicates a crucial role for continuous de novo spliced leader RNP assembly

ACKNOWLEDGEMENTS Some strains were provided by the CGC, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440). We would also like to thank Prof. Shohei Mitani,at the National Bioresource Project for the Experimental Animal ‘Nematode C. elegans’, Japan, for FX3079. We are grateful to Prof. Tom Blumenthal (University of Colorado, Boulder) for suggestions and support of this work; and to Kathrine Wood for her contribution to the initial stages of part of this work. Author contributions. L.P., G.P., R.F., N.H., J.P. and B.M. performed experiments; B.M., J.P. and B.C. designed and lead the study; B.M. and J.P. drafted the manuscript. All authors reviewed the manuscript. FUNDING Biotechnology and Biological Sciences Research Council (BBSRC) [Project grant BB/J007137/1]; Medical Research Council (MRC) Confidence in Concept 2014 - University of Aberdeen Award(MC PC 14114v.2); University of Aberdeen Elphinstone Scholarship (to R.F.) and TET Fund support through Adekunle Ajasin University, Nigeria (to R.F.). Funding for open access charge: Biotechnology and Biological Sciences Research Council and Medical Research Council.Peer reviewedPublisher PD

Parkinson's VPS35[D620N] retromer lysosomal dysfunction induces LRRK2 mediated lysosomal association of RILPL1 and TMEM55B

Primary data associated with the manuscript "Parkinson’s VPS35[D620N] mutation induces LRRK2 mediated lysosomal association of RILPL1 and TMEM55B" (Prosenjit Pal, Matthew Taylor, Pui Yiu Lam, Francesca Tonelli, Chloe A.Hecht, Pawel Lis, Raja S. Nirujogi,Toan K. Phung, Wondwossen M.Yeshaw, Ebsy Jaimon, Rotimi Fasimoye, Emily A. Dickie, MelanieWightman, Thomas Macartney, Suzanne R. Pfeffer and Dario R. Alessi) -- This is un updated version (after revision round #1) of Primary data associated with the manuscript "Parkinson’s VPS35[D620N] retromer lysosomal dysfunction induces LRRK2 mediated lysosomal association of RILPL1 and TMEM55B" (Prosenjit Pal, Matthew Taylor, Pui Yiu Lam, Francesca Tonelli, Chloe A. Hecht, Pawel Lis, Raja S. Nirujogi, Toan K. Phung, Emily A. Dickie, Mel Wightman, Thomas Macartney, Suzanne R. Pfeffer and Dario R. Alessi). These include raw immunoblotting data (annotated .tiff exports of Image Studio Lite Licor scans) with their quantitation (.xlsx files) and graphs with statistical analysis obtained using GraphPad Prism (.pzf files)

Targeted dephosphorylation of SMAD3 as an approach to impede TGFβ signaling

TGFβ (transforming growth factor-β) signaling is involved in a myriad of cellular processes and its dysregulation has been implicated in many human diseases, including fibrosis and cancer. TGFβ transcriptional responses are controlled by tail phosphorylation of transcription factors SMAD2 and SMAD3 (mothers against decapentaplegic homolog 2/3). Therefore, targeted dephosphorylation of phospho-SMAD3 could provide an innovative mechanism to block some TGFβ-induced transcriptional responses, such as the transcription of SERPINE-1, which encodes plasminogen activator inhibitor 1 (PAI-1). Here, by developing and employing a bifunctional molecule, BDPIC (bromoTAG-dTAG proximity inducing chimera), we redirected multiple phosphatases, tagged with bromoTAG, to dephosphorylate phospho-SMAD3, tagged with dTAG. Using CRISPR/Cas9 technology, we generated homozygousdouble knock-in A549 bromoTAG/bromoTAGPPM1H/dTAG/dTAG SMAD3 cells, in which the BDPIC-induced proximity between bromoTAG-PPM1H and dTAG-SMAD3 led to a robust dephosphorylation of dTAG-SMAD3 and a significant decrease in SERPINE-1 transcription. Our work demonstrates targeted dephosphorylation of phospho-proteins as an exciting modality for rewiring cell signaling

Development of BromoTag: A “bump-&-hole”-PROTAC system to induce potent, rapid, and selective degradation of tagged target proteins

Small-molecule-induced protein depletion technologies, also called inducible degrons, allow degradation of genetically engineered target proteins within cells and animals. Here, we design and develop the BromoTag, a new inducible degron system comprising a Brd4 bromodomain L387A variant as a degron tag that allows direct recruitment by heterobifunctional bumped proteolysis targeting chimeras (PROTACs) to hijack the VHL E3 ligase. We describe extensive optimization and structure-activity relationships of our bump-and-hole-PROTACs using a CRISPR knock-in cell line expressing model target BromoTag-Brd2 at endogenous levels. Collectively, our cellular and mechanistic data qualifies bumped PROTAC AGB1 as a potent, fast, and selective degrader of BromoTagged proteins, with a favorable pharmacokinetic profile in mice. The BromoTag adds to the arsenal of chemical genetic degradation tools allowing us to manipulate protein levels to interrogate the biological function and therapeutic potential in cells and in vivo