RNA tailing machinery drives amyloidogenic phase transition.

The RNA tailing machinery adds nucleotides to the 3'-end of RNA molecules that are implicated in various biochemical functions, including protein synthesis and RNA stability. Here, we report a role for the RNA tailing machinery as enzymatic modifiers of intracellular amyloidogenesis. A targeted RNA interference screen identified Terminal Nucleotidyl-transferase 4b (TENT4b/Papd5) as an essential participant in the amyloidogenic phase transition of nucleoli into solid-like Amyloid bodies. Full-length-and-mRNA sequencing uncovered starRNA, a class of unusually long untemplated RNA molecules synthesized by TENT4b. StarRNA consists of short rRNA fragments linked to long, linear mixed tails that operate as polyanionic stimulators of amyloidogenesis in cells and in vitro. Ribosomal intergenic spacer noncoding RNA (rIGSRNA) recruit TENT4b in intranucleolar foci to coordinate starRNA synthesis driving their amyloidogenic phase transition. The exoribonuclease RNA Exosome degrades starRNA and functions as a general suppressor of cellular amyloidogenesis. We propose that amyloidogenic phase transition is under tight enzymatic control by the RNA tailing and exosome axis.

Nucleolar RNA polymerase II drives ribosome biogenesis.

Proteins are manufactured by ribosomes-macromolecular complexes of protein and RNA molecules that are assembled within major nuclear compartments called nucleoli1,2. Existing models suggest that RNA polymerases I and III (Pol I and Pol III) are the only enzymes that directly mediate the expression of the ribosomal RNA (rRNA) components of ribosomes. Here we show, however, that RNA polymerase II (Pol II) inside human nucleoli operates near genes encoding rRNAs to drive their expression. Pol II, assisted by the neurodegeneration-associated enzyme senataxin, generates a shield comprising triplex nucleic acid structures known as R-loops at intergenic spacers flanking nucleolar rRNA genes. The shield prevents Pol I from producing sense intergenic noncoding RNAs (sincRNAs) that can disrupt nucleolar organization and rRNA expression. These disruptive sincRNAs can be unleashed by Pol II inhibition, senataxin loss, Ewing sarcoma or locus-associated R-loop repression through an experimental system involving the proteins RNaseH1, eGFP and dCas9 (which we refer to as 'red laser'). We reveal a nucleolar Pol-II-dependent mechanism that drives ribosome biogenesis, identify disease-associated disruption of nucleoli by noncoding RNAs, and establish locus-targeted R-loop modulation. Our findings revise theories of labour division between the major RNA polymerases, and identify nucleolar Pol II as a major factor in protein synthesis and nuclear organization, with potential implications for health and disease.

Yeast Fin1-PP1 dephosphorylates an Ipl1 substrate, Ndc80, to remove Bub1-Bub3 checkpoint proteins from the kinetochore during anaphase.

The spindle assembly checkpoint (SAC) prevents anaphase onset in response to chromosome attachment defects, and SAC silencing is essential for anaphase onset. Following anaphase onset, activated Cdc14 phosphatase dephosphorylates the substrates of cyclin-dependent kinase to facilitate anaphase progression and mitotic exit. In budding yeast, Cdc14 dephosphorylates Fin1, a regulatory subunit of protein phosphatase 1 (PP1), to enable kinetochore localization of Fin1-PP1. We previously showed that kinetochore-localized Fin1-PP1 promotes the removal of the SAC protein Bub1 from the kinetochore during anaphase. We report here that Fin1-PP1 also promotes kinetochore removal of Bub3, the Bub1 partner, but has no effect on another SAC protein Mad1. Moreover, the kinetochore localization of Bub1-Bub3 during anaphase requires Aurora B/Ipl1 kinase activity. We further showed that Fin1-PP1 facilitates the dephosphorylation of kinetochore protein Ndc80, a known Ipl1 substrate. This dephosphorylation reduces kinetochore association of Bub1-Bub3 during anaphase. In addition, we found that untimely Ndc80 dephosphorylation causes viability loss in response to tensionless chromosome attachments. These results suggest that timely localization of Fin1-PP1 to the kinetochore controls the functional window of SAC and is therefore critical for faithful chromosome segregation.

Local translation in nuclear condensate amyloid bodies.

Biomolecular condensates concentrate molecules to facilitate basic biochemical processes, including transcription and DNA replication. While liquid-like condensates have been ascribed various functions, solid-like condensates are generally thought of as amorphous sites of protein storage. Here, we show that solid-like amyloid bodies coordinate local nuclear protein synthesis (LNPS) during stress. On stimulus, translationally active ribosomes accumulate along fiber-like assemblies that characterize amyloid bodies. Mass spectrometry analysis identified regulatory ribosomal proteins and translation factors that relocalize from the cytoplasm to amyloid bodies to sustain LNPS. These amyloidogenic compartments are enriched in newly transcribed messenger RNA by Heat Shock Factor 1 (HSF1). Depletion of stress-induced ribosomal intergenic spacer noncoding RNA (rIGSRNA) that constructs amyloid bodies prevents recruitment of the nuclear protein synthesis machinery, abolishes LNPS, and impairs the nuclear HSF1 response. We propose that amyloid bodies support local nuclear translation during stress and that solid-like condensates can facilitate complex biochemical reactions as their liquid counterparts can.

A new layer of regulation of chromosomal passenger complex (CPC) translocation in budding yeast.

The conserved chromosomal passenger complex (CPC) consists of Ipl1Aurora-B, Sli15INCENP, Bir1Survivin, and Nbl1Borealin, and localizes at the kinetochore/centromere to correct kinetochore attachment errors and to prevent checkpoint silencing. After anaphase entry, the CPC moves from the kinetochore/centromere to the spindle. In budding yeast, CPC subunit Sli15 is phosphorylated by both cyclin-dependent kinase (CDK) and Ipl1 kinase. Following anaphase onset, activated Cdc14 phosphatase reverses Sli15 phosphorylation imposed by CDK to promote CPC translocation. Although abolished Sli15 phosphorylation imposed by Ipl1 also causes CPC translocation, the regulation of Ipl1-imposed Sli15 phosphorylation remains unclear. In addition to Sli15, Cdc14 also dephosphorylates Fin1, a regulatory subunit of protein phosphatase 1 (PP1), to enable kinetochore localization of Fin1-PP1. Here, we present evidence supporting the notion that kinetochore-localized Fin1-PP1 likely reverses Ipl1-imposed Sli15 phosphorylation to promote CPC translocation from the kinetochore/centromere to the spindle. Importantly, premature Fin1 kinetochore localization or phospho-deficient sli15 mutation causes checkpoint defects in response to tensionless attachments, resulting in chromosome missegregation. In addition, our data indicate that reversion of CDK- and Ipl1-imposed Sli15 phosphorylation shows an additive effect on CPC translocation. Together, these results reveal a previously unidentified pathway to regulate CPC translocation, which is important for accurate chromosome segregation.

A translational program that suppresses metabolism to shield the genome.

Translatome reprogramming is a primary determinant of protein levels during stimuli adaptation. This raises the question: what are the translatome remodelers that reprogram protein output to activate biochemical adaptations. Here, we identify a translational pathway that represses metabolism to safeguard genome integrity. A system-wide MATRIX survey identified the ancient eIF5A as a pH-regulated translation factor that responds to fermentation-induced acidosis. TMT-pulse-SILAC analysis identified several pH-dependent proteins, including the mTORC1 suppressor Tsc2 and the longevity regulator Sirt1. Sirt1 operates as a pH-sensor that deacetylates nuclear eIF5A during anaerobiosis, enabling the cytoplasmic export of eIF5A/Tsc2 mRNA complexes for translational engagement. Tsc2 induction inhibits mTORC1 to suppress cellular metabolism and prevent acidosis-induced DNA damage. Depletion of eIF5A or Tsc2 leads to metabolic re-initiation and proliferation, but at the expense of incurring substantial DNA damage. We suggest that eIF5A operates as a translatome remodeler that suppresses metabolism to shield the genome.

Nucleolar Sequestration: Remodeling Nucleoli Into Amyloid Bodies.

This year marks the 20th anniversary of the discovery that the nucleolus can temporarily immobilize proteins, a process known as nucleolar sequestration. This review reflects on the progress made to understand the physiological roles of nucleolar sequestration and the mechanisms involved in the immobilization of proteins. We discuss how protein immobilization can occur through a highly choreographed amyloidogenic program that converts the nucleolus into a large fibrous organelle with amyloid-like characteristics called the amyloid body (A-body). We propose a working model of A-body biogenesis that includes a role for low-complexity ribosomal intergenic spacer RNA (rIGSRNA) and a discrete peptide sequence, the amyloid-converting motif (ACM), found in many proteins that undergo immobilization. Amyloid bodies provide a unique model to study the multistep assembly of a membraneless compartment and may provide alternative insights into the pathological amyloidogenesis involved in neurological disorders.

The phosphorylation of a kinetochore protein Dam1 by Aurora B/Ipl1 kinase promotes chromosome bipolar attachment in yeast.

The interaction between chromosomes and spindle microtubules is essential for chromosome segregation. The kinetochore complex mediates this interaction. Previous studies indicate that the stability of kinetochore attachment is regulated by Aurora B/Ipl1 kinase and this regulation is conserved from yeast to mammalian cells. In budding yeast Saccharomyces cerevisiae, the ten-subunit Dam1/DASH complex bridges the interaction between kinetochores and microtubules, and some in vitro evidence indicates that the phosphorylation of Dam1 protein by Ipl1 kinase destabilizes this interaction. However, it is not clear if Dam1 phosphorylation is sufficient to regulate the stability of kinetochore attachment in vivo. Also, the significance of this regulation in response to chromosome detachment has not been fully investigated. Here we report that phospho-deficient dam1-3A mutants show stabilized kinetochore-microtubule attachment in vivo. This significantly delays the establishment of chromosome bipolar attachment after the disruption of kinetochore-microtubule interaction by a microtubule depolymerizing drug nocodazole. Moreover, dam1-3A cells show dramatic chromosome mis-segregation after treatment with nocodazole, presumably due to the combination of compromised bipolar attachment and premature spindle assembly checkpoint silencing in the mutant cells. Therefore, the regulation of Dam1 phosphorylation imposed by Ipl1 kinase is critical for faithful chromosome segregation.

Premature Silencing of the Spindle Assembly Checkpoint Is Prevented by the Bub1-H2A-Sgo1-PP2A Axis in Saccharomyces cerevisiae.

The spindle assembly checkpoint (SAC) monitors mistakes in kinetochore-microtubule interaction and its activation prevents anaphase entry. The SAC remains active until all chromosomes have achieved bipolar attachment which applies tension on kinetochores. Our previous data in budding yeast Saccharomyces cerevisiae show that Ipl1/Aurora B kinase and a centromere-associated protein, Sgo1, are required to prevent SAC silencing prior to tension generation, but we believe that this regulatory network is incomplete. Bub1 kinase is one of the SAC components, and Bub1-dependent H2A phosphorylation triggers centromere recruitment of Sgo1 by H2A in yeast and human cells. Although yeast cells lacking the kinase domain of Bub1 show competent SAC activation, we found that the mutant cells fail to maintain a prolonged checkpoint arrest in the presence of tensionless attachment. Mutation of the Bub1 phosphorylation site in H2A also results in premature SAC silencing in yeast cells. Previous data indicate that Sgo1 protein binds to PP2ARts1, and we found that rts1Δ mutants exhibited premature SAC silencing as well. We further revealed that sgo1 mutants with abolished binding to H2A or PP2ARts1 displayed premature SAC silencing. Together, our results suggest that, in budding yeast S. cerevisiae, the Bub1-H2A-Sgo1-PP2ARts1 axis prevents SAC silencing and helps prolonged checkpoint arrest prior to tension establishment at kinetochores.

Fin1-PP1 Helps Clear Spindle Assembly Checkpoint Protein Bub1 from Kinetochores in Anaphase.

The spindle assembly checkpoint (SAC) monitors chromosome attachment defects, and the assembly of SAC proteins at kinetochores is essential for its activation, but the SAC disassembly process remains unknown. We found that deletion of a 14-3-3 protein, Bmh1, or hyperactivation of Cdc14 early anaphase release (FEAR) allows premature SAC silencing in budding yeast, which depends on a kinetochore protein Fin1 that forms a complex with protein phosphatase PP1. Previous works suggest that FEAR-dependent Fin1 dephosphorylation promotes Bmh1-Fin1 dissociation, which enables kinetochore recruitment of Fin1-PP1. We found persistent kinetochore association of SAC protein Bub1 in fin1Δ mutants after anaphase entry. Therefore, we revealed a mechanism that clears SAC proteins from kinetochores. After anaphase entry, FEAR activation promotes kinetochore enrichment of Fin1-PP1, resulting in SAC disassembly at kinetochores. This mechanism is required for efficient SAC silencing after SAC is challenged, and untimely Fin1-kinetochore association causes premature SAC silencing and chromosome missegregation.