Global analysis of serine/threonine and tyrosine protein phosphatase catalytic subunit genes in Neurospora crassa reveals interplay between phosphatases and the p38 mitogen-activated protein kinase.

Protein phosphatases are integral components of the cellular signaling machinery in eukaryotes, regulating diverse aspects of growth and development. The genome of the filamentous fungus and model organism Neurospora crassa encodes catalytic subunits for 30 protein phosphatase genes. In this study, we have characterized 24 viable N. crassa phosphatase catalytic subunit knockout mutants for phenotypes during growth, asexual development, and sexual development. We found that 91% of the mutants had defects in at least one of these traits, whereas 29% possessed phenotypes in all three. Chemical sensitivity screens were conducted to reveal additional phenotypes for the mutants. This resulted in the identification of at least one chemical sensitivity phenotype for 17 phosphatase knockout mutants, including novel chemical sensitivities for two phosphatase mutants lacking a growth or developmental phenotype. Hence, chemical sensitivity or growth/developmental phenotype was observed for all 24 viable mutants. We investigated p38 mitogen-activated protein kinase (MAPK) phosphorylation profiles in the phosphatase mutants and identified nine potential candidates for regulators of the p38 MAPK. We demonstrated that the PP2C class phosphatase pph-8 (NCU04600) is an important regulator of female sexual development in N. crassa. In addition, we showed that the Δcsp-6 (ΔNCU08380) mutant exhibits a phenotype similar to the previously identified conidial separation mutants, Δcsp-1 and Δcsp-2, that lack transcription factors important for regulation of conidiation and the circadian clock

Mapping the mammalian ribosome quality control complex interactome using proximity labeling approaches.

Previous genetic and biochemical studies from Saccharomyces cerevisiae have identified a critical ribosome-associated quality control complex (RQC) that facilitates resolution of stalled ribosomal complexes. While components of the mammalian RQC have been examined in vitro, a systematic characterization of RQC protein interactions in mammalian cells has yet to be described. Here we utilize both proximity-labeling proteomic approaches, BioID and APEX, and traditional affinity-based strategies to both identify interacting proteins of mammalian RQC members and putative substrates for the RQC resident E3 ligase, Ltn1. Surprisingly, validation studies revealed that a subset of substrates are ubiquitylated by Ltn1 in a regulatory manner that does not result in subsequent substrate degradation. We demonstrate that Ltn1 catalyzes the regulatory ubiquitylation of ribosomal protein S6 kinase 1 and 2 (RPS6KA1, RPS6KA3). Further, loss of Ltn1 function results in hyperactivation of RSK1/2 signaling without impacting RSK1/2 protein turnover. These results suggest that Ltn1-mediated RSK1/2 ubiquitylation is inhibitory and establishes a new role for Ltn1 in regulating mitogen-activated kinase signaling via regulatory RSK1/2 ubiquitylation. Taken together, our results suggest that mammalian RQC interactions are difficult to observe and may be more transient than the homologous complex in S. cerevisiae and that Ltn1 has RQC-independent functions

Characterization of RNAi-defective mutants -- Mut13-2 and Mut20 -- in \u3ci\u3eChlamydomonas reinhardtii\u3c/i\u3e

RNA interference (RNAi) is a highly conserved and sequence-specific process for regulation of gene expression. At the heart of the RNAi machinery lie 21-24 nt small non-coding RNA molecules which are able to downregulate the expression of cognate sequences, by causing degradation or translational repression of mRNAs. These noncoding RNAs act via the effector RNA-induced silencing complex (RISC), which includes core proteins such as the Argonautes (AGOs). Two Chlamydomonas reinhardtii RNAi-defective mutants, Mut13-2 and Mut20, show deletions of various domains of the Tudor Staphylococcal Nuclease 1 (TSN1) gene. TSN1 has already been recognized as a component of the RNA-induced silencing complex in metazoans. However, the specific function of this protein in the RNAi pathway remains undefined. In this study we report that TSN1 is essential for RNAi in Chlamydomonas reinhardtii. Interestingly, the two examined mutants show a widely different profile of endogenous miRNAs. The small RNAs are almost completely depleted in the case of Mut20 but are only slightly reduced in level in Mut13-2. Successful complementation with an epitope-tagged TSN1 and recovery of RNAi-induced phenotypes was achieved in Mut13-2 but not in Mut20. Through immunofluorescence microscopy, the epitope-tagged TSN1 protein was found to be predominantly cytoplasmic, although some degree of nuclear localization was also observed, which supports its association with the RISC as reported in metazoans. Yet, coimmunoprecipitation assays with the epitope-tagged TSN1 did not show association with AGO3, a core component of the RISC in Chlamydomonas. It is tempting to hypothesize that TSN1 may play an accessory role in the assembly of the RISC, perhaps in the loading of small RNAs onto Argonautes. This interpretation would be consistent with the reduced level of small RNAs in the mutant backgrounds and the lack of high affinity interactions with AGO proteins. However, elucidating the exact role of TSN1 will require further investigation. Advisor: Heriberto Cerutt

Cellular Signaling Mechanisms in Neurospora crassa

Cellular signal transduction mechanisms are regulated at multiple different stages during an organism’s life cycle as well as life span. Environmental stress and starvation responses are extremely well coordinated by cell surface receptors and internal scaffolding molecules that are routing the signal to the effector proteins such as transcription factors. It is through these transcription factors that the cell will then regulate gene expression. With the help of this study, my co-authors and me have tried to elucidate a signal transduction network, which explains various facets of cellular signaling in the model filamentous fungus – Neurospora crassa. The first chapter has elucidated the role of serine/threonine and tyrosine phosphatases in growth and development in Neurospora. In addition, the chapter also shows that deletion of certain phosphatases lead to sensitivities to chemicals inducing osmotic stress, oxidative stress, cytoskeletal defects or ROS accumulation. Nine phosphatase mutants are also listed to have elevated levels of the active phosphorylated form of p38 mitogen activated protein kinase (OS-2 in Neurospora) which is a critical regulator for counteraction to osmo-stress as well as an important regulator of female sexual development. Two other interesting genes – NCU04600 (pph-8) and NCU08380 (csp-6) are also described in this chapter for their unique phenotypes. The second chapter deals with the role of the important scaffolding protein and RACK1 homolog – CPC-2 in regulation of amino acid starvation mechanisms in Neurospora known as cross pathway control. CPC-2 is found to regulate the bZIP transcription factor – CPC-1 via modulation of post-translational modifications on CPC-1 during amino acid starvation. This transcription factor is in turn, integral towards de-repression of amino acid biosynthetic genes under amino acid starvation conditions. This chapter provides mechanistic details on how CPC-2 is able to regulate CPC-1 protein and thereby affect cross pathway control. In chapter III, the focus is shifted towards translational regulation via heterotrimeric G proteins and the guanine-exchange factor RIC-8 to build up a novel finding revealing that G proteins and RIC-8 are an integral part of the ribosome. Elevated phospho-eIF2α levels in the G protein and ric-8 mutants suggest that global translation is greatly reduced in these strains. Poly-RNA-seq analyses of gna-1, gnb-1 and ric-8 mutants reveal certain ribosomal proteins as well as elongation factors and two serine/threonine kinases – stk-18 and stk-43 are greatly affected in polysomal co-migration by these gene deletions. In addition, deletion of ric-8 leads to loss of PKC protein from the polysomes, which suggests critical translational control of PKC via RIC-8. This thesis has thus aimed to expand on current knowledge on these abovementioned topics and laid the groundwork for future advances in understanding these cellular signaling mechanisms in Neurospora crassa

Genetic relationships between the RACK1 homolog cpc-2 and heterotrimeric G protein subunit genes in Neurospora crassa.

Receptor for Activated C Kinase-1 (RACK1) is a multifunctional eukaryotic scaffolding protein with a seven WD repeat structure. Among their many cellular roles, RACK1 homologs have been shown to serve as alternative Gβ subunits during heterotrimeric G protein signaling in many systems. We investigated genetic interactions between the RACK1 homolog cpc-2, the previously characterized Gβ subunit gnb-1 and other G protein signaling components in the multicellular filamentous fungus Neurospora crassa. Results from cell fractionation studies and from fluorescent microscopy of a strain expressing a CPC-2-GFP fusion protein revealed that CPC-2 is a cytoplasmic protein. Genetic epistasis experiments between cpc-2, the three Gα genes (gna-1, gna-2 and gna-3) and gnb-1 demonstrated that cpc-2 is epistatic to gna-2 with regards to basal hyphae growth rate and aerial hyphae height, while deletion of cpc-2 mitigates the increased macroconidiation on solid medium observed in Δgnb-1 mutants. Δcpc-2 mutants inappropriately produce conidiophores during growth in submerged culture and mutational activation of gna-3 alleviates this defect. Δcpc-2 mutants are female-sterile and fertility could not be restored by mutational activation of any of the three Gα genes. With the exception of macroconidiation on solid medium, double mutants lacking cpc-2 and gnb-1 exhibited more severe defects for all phenotypic traits, supporting a largely synergistic relationship between GNB-1 and CPC-2 in N. crassa

Genetic relationships between the RACK1 homolog cpc-2 and heterotrimeric G protein subunit genes in Neurospora crassa.

Receptor for Activated C Kinase-1 (RACK1) is a multifunctional eukaryotic scaffolding protein with a seven WD repeat structure. Among their many cellular roles, RACK1 homologs have been shown to serve as alternative Gβ subunits during heterotrimeric G protein signaling in many systems. We investigated genetic interactions between the RACK1 homolog cpc-2, the previously characterized Gβ subunit gnb-1 and other G protein signaling components in the multicellular filamentous fungus Neurospora crassa. Results from cell fractionation studies and from fluorescent microscopy of a strain expressing a CPC-2-GFP fusion protein revealed that CPC-2 is a cytoplasmic protein. Genetic epistasis experiments between cpc-2, the three Gα genes (gna-1, gna-2 and gna-3) and gnb-1 demonstrated that cpc-2 is epistatic to gna-2 with regards to basal hyphae growth rate and aerial hyphae height, while deletion of cpc-2 mitigates the increased macroconidiation on solid medium observed in Δgnb-1 mutants. Δcpc-2 mutants inappropriately produce conidiophores during growth in submerged culture and mutational activation of gna-3 alleviates this defect. Δcpc-2 mutants are female-sterile and fertility could not be restored by mutational activation of any of the three Gα genes. With the exception of macroconidiation on solid medium, double mutants lacking cpc-2 and gnb-1 exhibited more severe defects for all phenotypic traits, supporting a largely synergistic relationship between GNB-1 and CPC-2 in N. crassa