In this dissertation, I use the early embryo of the nematode Caenorhabditis elegans to probe the relationships between kinetochore formation and spindle checkpoint signaling. The spindle checkpoint is a conserved signaling pathway that delays cell cycle progression in mitosis until all chromosomes have properly attached to microtubules emanating from opposing spindle poles. To trigger checkpoint activation, I undertook a strategy of inhibiting centriole duplication, using RNAi specific for components of the centriole duplication machinery. Such depletions result in bipolar spindles in the first mitotic division (serving as useful internal controls), while leading to monopolar spindle formation in the second and third divisions. Monopolar spindles are known to trigger checkpoint activation, as kinetochores lack both full attachment and tension. By performing live imaging video microscopy on these embryos, I was able to quantitatively assess checkpoint status by timing cell cycle progression. I found that cells with monopolar spindles exhibited cell cycle delays that were dependent on each of the conserved checkpoint proteins in C. elegans. Kinetochores are proteinaceus organelles that connect DNA to microtubules, and are essential for chromosome segregation in mitosis. Using the timing assay described above, I set out to comprehensively determine the contribution of kinetochore proteins for checkpoint signaling, and found that these proteins could be placed into one of three categories based on their phenotypes in the timing assay; required for checkpoint signaling, not required, or those that trigger checkpoint activation when depleted. Checkpoint activation is correlated with enrichment of specific components of the pathway on unattached kinetochores. To correlate checkpoint protein recruitment with the functional analysis of checkpoint signaling, I generated worm strains stably expressing fluorescently tagged checkpoint proteins Mad2^{MDF⁻², Mad3SAN⁻¹, and BUB-3, and systematically determined the requirements for their localization and enrichment at kinetochores. I also used these assays to characterize the roles of the recently discovered kinetochore protein SPDL-1 in checkpoint activation and signaling. Taken together, my results indicate that checkpoint activation is coordinately directed by three key kinetochore components - the NDC-80 complex, the Rod/Zwilch/Zw10 complex, and BUB -1 - that are targeted independently of one another by the outer kinetochore scaffold protein KNL-1. Surprisingly, I found that Mad3SAN⁻¹, unlike other checkpoint proteins, does not enrich at unattached kinetochores, and that subtly elevating Mad2MDF⁻², levels bypasses the requirement for BUB-3 and Mad3SAN⁻¹ in kinetochore-dependent checkpoint activation. I propose that a core kinetochore-generated Mad1MDF⁻¹/super>/ Mad2MDF⁻² signal is integrated with a largely independent cytoplasmic Mad3SAN⁻¹/BUB-3-based signal to coordinately regulate checkpoint activation, possibly in response to different checkpoint-activating states. I discuss the rationale for the partitioning of the checkpoint signaling pathway into two branches, and the newly elucidated relationships between kinetochore assembly and checkpoint signaling}

Human pluripotent stem cells (hPSCs) require precise control of post-transcriptional RNA networks to maintain proliferation and survival. Using enhanced UV crosslinking and immunoprecipitation (eCLIP), we identify RNA targets of the IMP/IGF2BP family of RNA-binding proteins in hPSCs. At the broad region and binding site levels, IMP1 and IMP2 show reproducible binding to a large and overlapping set of 3' UTR-enriched targets. RNA Bind-N-seq applied to recombinant full-length IMP1 and IMP2 reveals CA-rich motifs that are enriched in eCLIP-defined binding sites. We observe that IMP1 loss in hPSCs recapitulates IMP1 phenotypes, including a reduction in cell adhesion and increase in cell death. For cell adhesion, we find IMP1 maintains levels of integrin mRNA specifically regulating RNA stability of ITGB5 in hPSCs. Additionally, we show that IMP1 can be linked to hPSC survival via direct target BCL2. Thus, transcriptome-wide binding profiles identify hPSC targets modulating well-characterized IMP1 roles.