Elongational stalling activates mitoribosome-associated quality control

The human mitochondrial ribosome (mitoribosome) and associated proteins regulate the synthesis of 13 essential subunits of the oxidative phosphorylation complexes. We report the discovery of a mitoribosome-associated quality control pathway that responds to interruptions during elongation, and we present structures at 3.1- to 3.3-angstrom resolution of mitoribosomal large subunits trapped during ribosome rescue. Release factor homolog C12orf65 (mtRF-R) and RNA binding protein C6orf203 (MTRES1) eject the nascent chain and peptidyl transfer RNA (tRNA), respectively, from stalled ribosomes. Recruitment of mitoribosome biogenesis factors to these quality control intermediates suggests additional roles for these factors during mitoribosome rescue. We also report related cryo–electron microscopy structures (3.7 to 4.4 angstrom resolution) of elongating mitoribosomes bound to tRNAs, nascent polypeptides, the guanosine triphosphatase elongation factors mtEF-Tu and mtEF-G1, and the Oxa1L translocase.

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The Putative RNA-Binding Protein Nrp1 Promotes the Loading of Kinesin-14/Klp2 to the Mitotic Spindle and Is Sequestered Into Heat-Induced Protein Aggregates in Fission Yeast

10.20944/preprints202103.0452.v1 ◽

2021 ◽

Author(s):

Masashi Yukawa ◽

Mitsuki Ohishi ◽

Yusuke Yamada ◽

Takashi Toda

Keyword(s):

Fission Yeast ◽

Rna Binding ◽

Binding Protein ◽

Rna Binding Protein ◽

Protein Aggregates ◽

Associated Proteins ◽

Spindle Microtubules ◽

The Stability ◽

And Function ◽

Post Transcriptional Regulation

Cells form a bipolar spindle during mitosis to ensure accurate chromosome segregation. Proper spindle architecture is established by a set of kinesin motors and microtubule-associated proteins. In most eukaryotes, kinesin-5 motors are essential for this process, and genetic or chemical inhibition of their activity leads to the emergence of monopolar spindles and cell death. However, these deficiencies can be rescued by simultaneous inactivation of kinesin-14 motors, as they counteract kinesin-5. We conducted detailed genetic analyses in fission yeast to understand the mechanisms driving spindle assembly in the absence of kinesin-5. Here we show that deletion of the nrp1 gene, which encodes a putative RNA-binding protein with unknown function, can rescue temperature sensitivity caused by cut7-22, a fission yeast kinesin-5 mutant. Interestingly, kinesin-14/Klp2 levels on the spindles in the cut7 mutants were significantly reduced by the nrp1 deletion, although the total levels of Klp2 and the stability of spindle microtubules remained unaffected. Moreover, RNA-binding motifs of Nrp1 are essential for its cytoplasmic localization and function. We have also found that a portion of Nrp1 is spatially and functionally sequestered by chaperone-based protein aggregates upon mild heat stress and limits cell division at high temperatures. We propose that Nrp1 might be involved in post-transcriptional regulation through its RNA-binding ability to promote the loading of Klp2 on the spindle microtubules.

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The landscape of translational stall sites in bacteria revealed by monosome and disome profiling

RNA ◽

10.1261/rna.078188.120 ◽

2021 ◽

pp. rna.078188.120

Author(s):

Tomoya Fujita ◽

Takeshi Yokoyama ◽

Mikako Shirouzu ◽

Hideki Taguchi ◽

Takuhiro Ito ◽

...

Keyword(s):

Electron Microscopy ◽

Low Frequency ◽

Ribosome Profiling ◽

Reporter Assay ◽

Ribosome Stalling ◽

Peptidyl Transfer ◽

Cryo Electron Microscopy ◽

Nascent Chain ◽

Biochemical Validation ◽

Trna Sequencing

Ribosome pauses are associated with various cotranslational events and determine the fate of mRNAs and proteins. Thus, the identification of precise pause sites across the transcriptome is desirable; however, the landscape of ribosome pauses in bacteria remains ambiguous. Here, we harness monosome and disome (or collided ribosome) profiling strategies to survey ribosome pause sites in Escherichia coli. Compared to eukaryotes, ribosome collisions in bacteria showed remarkable differences: a low frequency of disomes at stop codons, collisions occurring immediately after 70S assembly on start codons, and shorter queues of ribosomes trailing upstream. The pause sites corresponded with the biochemical validation by integrated nascent chain profiling (iNP) to detect polypeptidyl-tRNA, an elongation intermediate. Moreover, the subset of those sites showed puromycin resistance, presenting slow peptidyl transfer. Among the identified sites, the ribosome pause at Asn586 of ycbZ was validated by biochemical reporter assay, tRNA sequencing (tRNA-Seq), and cryo-electron microscopy (cryo-EM) experiments. Our results provide a useful resource for ribosome stalling sites in bacteria

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Full-length NF-κB repressing factor contains an XRN2 binding domain

Biochemical Journal ◽

10.1042/bcj20190733 ◽

2020 ◽

Vol 477 (4) ◽

pp. 773-786

Author(s):

Jana Alexandrova ◽

David Piñeiro ◽

Rebekah Jukes-Jones ◽

Ryan Mordue ◽

Mark Stoneley ◽

...

Keyword(s):

Ribosomal Rna ◽

Ribosome Biogenesis ◽

Rna Binding ◽

Rna Binding Protein ◽

Binding Domain ◽

Full Length ◽

Start Codon ◽

Rrna Processing ◽

Binding Partners ◽

Associated Proteins

NF-κB repressing factor (NKRF) was recently identified as an RNA binding protein that together with its associated proteins, the 5′–3′ exonuclease XRN2 and the helicase DHX15, is required to process the precursor ribosomal RNA. XRN2 is a multi-functional ribonuclease that is also involved in processing mRNAs, tRNAs and lncRNAs. The activity and stability of XRN2 are controlled by its binding partners, PAXT-1, CDKN2AIP and CDKN2AIPNL. In each case, these proteins interact with XRN2 via an XRN2 binding domain (XTBD), the structure and mode of action of which is highly conserved. Rather surprisingly, although NKRF interacts directly with XRN2, it was not predicted to contain such a domain, and NKRF's interaction with XRN2 was therefore unexplained. We have identified an alternative upstream AUG start codon within the transcript that encodes NKRF and demonstrate that the full-length form of NKRF contains an XTBD that is conserved across species. Our data suggest that NKRF is tethered in the nucleolus by binding directly to rRNA and that the XTBD in the N-terminal extension of NKRF is essential for the retention of XRN2 in this sub-organelle. Thus, we propose NKRF regulates the early steps of pre-rRNA processing during ribosome biogenesis by controlling the spatial distribution of XRN2 and our data provide further support for the XTBD as an XRN2 interacting motif.

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The E3 ubiquitin ligase and RNA-binding protein ZNF598 orchestrates ribosome quality control of premature polyadenylated mRNAs

Nature Communications ◽

10.1038/ncomms16056 ◽

2017 ◽

Vol 8 (1) ◽

Cited By ~ 76

Author(s):

Aitor Garzia ◽

Seyed Mehdi Jafarnejad ◽

Cindy Meyer ◽

Clément Chapat ◽

Tasos Gogakos ◽

...

Keyword(s):

Quality Control ◽

Ubiquitin Ligase ◽

Rna Binding ◽

Binding Protein ◽

Rna Binding Protein ◽

E3 Ubiquitin Ligase

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Cryo-EM structure of a neuronal functional amyloid implicated in memory persistence in Drosophila

Science ◽

10.1126/science.aba3526 ◽

2020 ◽

Vol 367 (6483) ◽

pp. 1230-1234 ◽

Cited By ~ 33

Author(s):

Ruben Hervas ◽

Michael J. Rau ◽

Younshim Park ◽

Wenjuan Zhang ◽

Alexey G. Murzin ◽

...

Keyword(s):

Electron Microscopy ◽

Amino Acid ◽

Rna Binding ◽

Rna Binding Protein ◽

Fundamental Question ◽

Hydrophobic Core ◽

Filament Formation ◽

Cytoplasmic Polyadenylation ◽

Cryo Electron Microscopy ◽

Memory Persistence

How long-lived memories withstand molecular turnover is a fundamental question. Aggregates of a prion-like RNA-binding protein, cytoplasmic polyadenylation element–binding (CPEB) protein, is a putative substrate of long-lasting memories. We isolated aggregated Drosophila CPEB, Orb2, from adult heads and determined its activity and atomic structure, at 2.6-angstrom resolution, using cryo–electron microscopy. Orb2 formed ~75-nanometer-long threefold-symmetric amyloid filaments. Filament formation transformed Orb2 from a translation repressor to an activator and “seed” for further translationally active aggregation. The 31–amino acid protofilament core adopted a cross-β unit with a single hydrophilic hairpin stabilized through interdigitated glutamine packing. Unlike the hydrophobic core of pathogenic amyloids, the hydrophilic core of Orb2 filaments suggests how some neuronal amyloids could be a stable yet regulatable substrate of memory.

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A Functional Interaction between the Evolutionarily Conserved Zinc Finger Poly(A) RNA‐binding Protein, Nab2, and the Exosome Links mRNA 3′‐End Processing/Export with mRNA Quality Control

The FASEB Journal ◽

10.1096/fasebj.23.1_supplement.666.1 ◽

2009 ◽

Vol 23 (S1) ◽

Author(s):

Milo Fasken ◽

Anita H Corbett

Keyword(s):

Quality Control ◽

Zinc Finger ◽

Rna Binding ◽

Binding Protein ◽

Rna Binding Protein ◽

Functional Interaction ◽

Evolutionarily Conserved

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The human RNA-binding protein RBFA promotes the maturation of the mitochondrial ribosome

Biochemical Journal ◽

10.1042/bcj20170256 ◽

2017 ◽

Vol 474 (13) ◽

pp. 2145-2158 ◽

Cited By ~ 15

Author(s):

Agata Rozanska ◽

Ricarda Richter-Dennerlein ◽

Joanna Rorbach ◽

Fei Gao ◽

Richard J. Lewis ◽

...

Keyword(s):

Rna Binding ◽

Binding Protein ◽

Rna Binding Protein ◽

Ribosomal Subunit ◽

Small Subunit ◽

12S Rrna ◽

Mitochondrial Rna ◽

Mitochondrial Ribosomes ◽

Mitochondrial Ribosome ◽

Rrna Species

Accurate assembly and maturation of human mitochondrial ribosomes is essential for synthesis of the 13 polypeptides encoded by the mitochondrial genome. This process requires the correct integration of 80 proteins, 1 mt (mitochondrial)-tRNA and 2 mt-rRNA species, the latter being post-transcriptionally modified at many sites. Here, we report that human ribosome-binding factor A (RBFA) is a mitochondrial RNA-binding protein that exerts crucial roles in mitoribosome biogenesis. Unlike its bacterial orthologue, RBFA associates mainly with helices 44 and 45 of the 12S rRNA in the mitoribosomal small subunit to promote dimethylation of two highly conserved consecutive adenines. Characterization of RBFA-depleted cells indicates that this dimethylation is not a prerequisite for assembly of the small ribosomal subunit. However, the RBFA-facilitated modification is necessary for completing mt-rRNA maturation and regulating association of the small and large subunits to form a functional monosome implicating RBFA in the quality control of mitoribosome formation.

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The Putative RNA-Binding Protein Dri1 Promotes the Loading of Kinesin-14/Klp2 to the Mitotic Spindle and Is Sequestered into Heat-Induced Protein Aggregates in Fission Yeast

International Journal of Molecular Sciences ◽

10.3390/ijms22094795 ◽

2021 ◽

Vol 22 (9) ◽

pp. 4795

Author(s):

Masashi Yukawa ◽

Mitsuki Ohishi ◽

Yusuke Yamada ◽

Takashi Toda

Keyword(s):

Fission Yeast ◽

Rna Binding ◽

Binding Protein ◽

Rna Binding Protein ◽

Protein Aggregates ◽

Associated Proteins ◽

Spindle Microtubules ◽

The Stability ◽

And Function ◽

Post Transcriptional Regulation

Cells form a bipolar spindle during mitosis to ensure accurate chromosome segregation. Proper spindle architecture is established by a set of kinesin motors and microtubule-associated proteins. In most eukaryotes, kinesin-5 motors are essential for this process, and genetic or chemical inhibition of their activity leads to the emergence of monopolar spindles and cell death. However, these deficiencies can be rescued by simultaneous inactivation of kinesin-14 motors, as they counteract kinesin-5. We conducted detailed genetic analyses in fission yeast to understand the mechanisms driving spindle assembly in the absence of kinesin-5. Here, we show that deletion of the dri1 gene, which encodes a putative RNA-binding protein, can rescue temperature sensitivity caused by cut7-22, a fission yeast kinesin-5 mutant. Interestingly, kinesin-14/Klp2 levels on the spindles in the cut7 mutants were significantly reduced by the dri1 deletion, although the total levels of Klp2 and the stability of spindle microtubules remained unaffected. Moreover, RNA-binding motifs of Dri1 are essential for its cytoplasmic localization and function. We have also found that a portion of Dri1 is spatially and functionally sequestered by chaperone-based protein aggregates upon mild heat stress and limits cell division at high temperatures. We propose that Dri1 might be involved in post-transcriptional regulation through its RNA-binding ability to promote the loading of Klp2 on the spindle microtubules.

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RNA Binding Protein TAF15 Suppresses Toxicity in a Yeast Model of FUS Proteinopathy

10.21203/rs.3.rs-437201/v1 ◽

2021 ◽

Author(s):

Elliott Hayden ◽

Aicha Kebe ◽

Shuzhen Chen ◽

Abagail Chumley ◽

Chenyi Xia ◽

...

Keyword(s):

Rna Binding ◽

Binding Protein ◽

Rna Binding Protein ◽

Stress Granules ◽

Rna Recognition Motif ◽

Body Formation ◽

P Bodies ◽

Associated Proteins ◽

Rnp Granules ◽

Yeast Model

Abstract Mutations in Fused in Sarcoma (FUS), an RNA binding protein that functions in multiple steps in gene expression regulation and RNA processing, are known to cause familial amyotrophic lateral sclerosis (ALS). Since this discovery, mutations in several other RNA binding proteins (RBPs) have also been linked to ALS. Some of these ALS-associated RBPs have been shown to colocalize with ribonucleoprotein (RNP) granules such as stress granules and processing bodies (p-bodies). Characterization of ALS-associated proteins, their mis-localization, aggregation and toxicity in cellular and animal models have provided critical insights in disease. More and more evidence has emerged supporting a hypothesis that impaired clearance, inappropriate assembly, and dysregulation of RNP granules play a role in ALS. Through genome-scale overexpression screening of a yeast model of FUS toxicity, we found that TAF15, a human RBP with a similar protein domain structure and belonging to the same FET protein family as FUS, suppresses FUS toxicity. The suppressor effect of TAF15 is specific to FUS and not found in other yeast models of neurodegenerative disease-associated proteins. We showed that the RNA recognition motif (RRM) of TAF15 is required for its rescue of FUS toxicity. Furthermore, FUS and TAF15 physically interact, and the C-terminus of TAF15 is required for both the physical protein-protein interaction and its protection against FUS toxicity. Finally, while FUS induces and colocalizes with both stress granules and p-bodies, TAF15 only induces and colocalizes with p-bodies. Importantly, co-expression of FUS and TAF15 induces more p-bodies than individually expressing each gene alone, and FUS toxicity is exacerbated in yeast that is deficient in p-body formation. Overall, our findings suggest a role of p-body formation in the suppression of FUS toxicity by TAF15.

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