Across a variety of Mendelian disorders, ∼50–75% of patients do not receive a genetic diagnosis by exome sequencing indicating disease-causing variants in non-coding regions. Although genome sequencing in principle reveals all genetic variants, their sizeable number and poorer annotation make prioritization challenging. Here, we demonstrate the power of transcriptome sequencing to molecularly diagnose 10% (5 of 48) of mitochondriopathy patients and identify candidate genes for the remainder. We find a median of one aberrantly expressed gene, five aberrant splicing events and six mono-allelically expressed rare variants in patient-derived fibroblasts and establish disease-causing roles for each kind. Private exons often arise from cryptic splice sites providing an important clue for variant prioritization. One such event is found in the complex I assembly factor TIMMDC1 establishing a novel disease-associated gene. In conclusion, our study expands the diagnostic tools for detecting non-exonic variants and provides examples of intronic loss-of-function variants with pathological relevance.

We have investigated the function of mitotic kinesin-like protein (MKlp) 2, a kinesin localized to the central spindle, and demonstrate that its depletion results in a failure of cleavage furrow ingression and cytokinesis, and disrupts localization of polo-like kinase 1 (Plk1). MKlp2 is a target for Plk1, and phosphorylated MKlp2 binds to the polo box domain of Plk1. Plk1 also binds directly to microtubules and targets to the central spindle via its polo box domain, and this interaction controls the activity of Plk1 toward MKlp2. An antibody to the neck region of MKlp2 that prevents phosphorylation of MKlp2 by Plk1 causes a cytokinesis defect when introduced into cells. We propose that phosphorylation of MKlp2 by Plk1 is necessary for the spatial restriction of Plk1 to the central spindle during anaphase and telophase, and the complex of these two proteins is required for cytokinesis.

44Across a large variety of Mendelian disorders, ~50-75% of patients do not receive a 45 genetic diagnosis by whole exome sequencing indicative of underlying disease-causing 46 variants in non-coding regions. In contrast, whole genome sequencing facilitates the 47 discovery of all genetic variants, but their sizeable number, coupled with a poor 48 understanding of the non-coding genome, makes their prioritization challenging. Here, we 49 demonstrate the power of transcriptome sequencing to provide a confirmed genetic 50 diagnosis for 10% (5 of 48) of undiagnosed mitochondrial disease patients and identify 51 strong candidate genes for patients remaining without diagnosis. We found a median of 1 52 aberrantly expressed gene, 5 aberrant splicing events, and 6 mono-allelically expressed 53 rare variants in patient-derived fibroblasts and established disease-causing roles for each 54 kind. Private exons often arose from sites that are weakly spliced in other individuals, 55providing an important clue for future variant prioritization. One such intronic exon-56 creating variant was found in three unrelated families in the complex I assembly factor 57 TIMMDC1, which we consequently established as a novel disease-associated gene. In 58 conclusion, our study expands the diagnostic tools for detecting non-exonic variants of 59Mendelian disorders and provides examples of intronic loss-of-function variants with 60 pathological relevance. 61Despite the revolutionizing impact of whole exome sequencing (WES) on the molecular 62 genetics of Mendelian disorders, ~50-75% of the patients do not receive a genetic diagnosis after 63 WES [1][2][3][4][5][6] . The disease-causing variants might be detected by WES but remain as variants of 64 unknown significance (VUS, Methods) or they are missed due to the inability to prioritize them. 65Many of these VUS are synonymous or non-coding variants that may affect RNA abundance or 66 isoform but cannot be prioritized due to the poor understanding of regulatory sequence to date 67 compared to coding sequence. Furthermore, WES covers only the 2% exonic regions of the 68 genome. Accordingly, it is mostly blind to regulatory variants in non-coding regions that could 69 affect RNA sequence and abundance. While the limitation of genome coverage is overcome by 70 whole genome sequencing (WGS), prioritization and interpretation of variants identified by 71 WGS is in turn limited by their amount [7][8][9] . 72With RNA sequencing (RNA-seq), limitations of the sole genetic information can be 73 complemented by directly probing variations in RNA abundance and in RNA sequence, 74 including allele-specific expression and splice isoforms. At least three extreme situations can be 75 directly interpreted to prioritize candidate disease-causing genes for a rare disorder. First, the 76 expression level of a gene can lie outside its physiological range. Genes with expression outside 77 their physical range can be identified as expression outliers, often using a stringent cutoff on 78 expression variat...

Spatial and temporal coordination of polo-like kinase 1 (Plk1) activity is necessary for mitosis and cytokinesis, and this is achieved through binding to phosphorylated docking proteins with distinct subcellular localizations. Although cyclin-dependent kinase 1 (Cdk1) creates these phosphorylated docking sites in metaphase, a general principle that explains how Plk1 activity is controlled in anaphase after Cdk1 inactivation is lacking. Here, we show that the microtubule-associated protein regulating cytokinesis (PRC1) is an anaphase-specific binding partner for Plk1, and that this interaction is required for cytokinesis. In anaphase, Plk1 creates its own docking site on PRC1, whereas in metaphase Cdk1 phosphorylates PRC1 adjacent to this docking site and thereby prevents binding of Plk1. Mutation of these Cdk1-sites results in a form of PRC1 that prematurely recruits Plk1 to the spindle during prometaphase and blocks mitotic progression. The activation state of Cdk1, therefore, controls the switch of Plk1 localization from centrosomes and kinetochores during metaphase, to the central spindle during anaphase.

The Golgi apparatus has long been suggested to be important for directing secretion to specific sites on the plasma membrane in response to extracellular signaling events. However, the mechanisms by which signaling events are coordinated with Golgi apparatus function remain poorly understood. Here, we identify a scaffolding function for the Golgi matrix protein GM130 that sheds light on how such signaling events may be regulated. We show that the mammalian Ste20 kinases YSK1 and MST4 target to the Golgi apparatus via the Golgi matrix protein GM130. In addition, GM130 binding activates these kinases by promoting autophosphorylation of a conserved threonine within the T-loop. Interference with YSK1 function perturbs perinuclear Golgi organization, cell migration, and invasion into type I collagen. A biochemical screen identifies 14-3-3ζ as a specific substrate for YSK1 that localizes to the Golgi apparatus, and potentially links YSK1 signaling at the Golgi apparatus with protein transport events, cell adhesion, and polarity complexes important for cell migration.

Rab-family GTPases are conserved regulators of membrane trafficking that cycle between inactive GDP-bound and activated GTP-bound states. A key determinant of Rab function is the lifetime of the GTP-bound state. As Rabs have a low intrinsic rate of GTP hydrolysis, this process is under the control of GTP-hydrolysis-activating proteins (GAPs). Due to the large number of Rabs and GAPs that are encoded by the human genome, it has proven difficult to assign specific functional relationships to these proteins. Here, we identify a Rab5-specific GAP (RabGAP-5), and show that RN-Tre (previously described as a Rab5 GAP) acts on Rab41. RabGAP-5 overexpression triggers a loss of the Rab5 effector EEA1 from endosomes and blocks endocytic trafficking. By contrast, depletion of RabGAP-5 results in increased endosome size, more endosome-associated EEA1, and disrupts the trafficking of EGF and LAMP1. RabGAP-5 therefore limits the amount of activated Rab5, and thereby regulates trafficking through endosomes.

Membrane traffic between the endoplasmic reticulum (ER) and Golgi apparatus and through the Golgi apparatus is a highly regulated process controlled by members of the rab GTPase family. The GTP form of rab1 regulates ER to Golgi transport by interaction with the vesicle tethering factor p115 and the cis-Golgi matrix protein GM130, also part of a complex with GRASP65 important for the organization of cis-Golgi cisternae. Here, we find that a novel coiled-coil protein golgin-45 interacts with the medial-Golgi matrix protein GRASP55 and the GTP form of rab2 but not other Golgi rab proteins. Depletion of golgin-45 disrupts the Golgi apparatus and causes a block in secretory protein transport. These results demonstrate that GRASP55 and golgin-45 form a rab2 effector complex on medial-Golgi essential for normal protein transport and Golgi structure.

GRASP65, a structural protein of the Golgi apparatus, has been linked to the sensing of Golgi structure and the integration of this information with the control of mitotic entry in the form of a Golgi checkpoint. We show that Cdk1-cyclin B is the major kinase phosphorylating GRASP65 in mitosis, and that phosphorylated GRASP65 interacts with the polo box domain of the polo-like kinase Plk1. GRASP65 is phosphorylated in its C-terminal domain at four consensus sites by Cdk1-cyclin B, and mutation of these residues to alanine essentially abolishes both mitotic phosphorylation and Plk1 binding. Expression of the wildtype GRASP65 C-terminus but not the phosphorylation defective mutant in normal rat kidney cells causes a delay but not the block in mitotic entry expected if this were a true cell cycle checkpoint. These findings identify a Plk1-dependent signalling mechanism potentially linking Golgi structure and cell cycle control, but suggest that this may not be a cell cycle checkpoint in the classical sense.