piRNA processing by a trimeric Schlafen-domain nuclease.

Transposable elements are genomic parasites that expand within and spread between genomes1. PIWI proteins control transposon activity, notably in the germline2,3. These proteins recognize their targets through small RNA co-factors named PIWI-interacting RNAs (piRNAs), making piRNA biogenesis a key specificity-determining step in this crucial genome immunity system. Although the processing of piRNA precursors is an essential step in this process, many of the molecular details remain unclear. Here, we identify an endoribonuclease, precursor of 21U RNA 5'-end cleavage holoenzyme (PUCH), that initiates piRNA processing in the nematode Caenorhabditis elegans. Genetic and biochemical studies show that PUCH, a trimer of Schlafen-like-domain proteins (SLFL proteins), executes 5'-end piRNA precursor cleavage. PUCH-mediated processing strictly requires a 7-methyl-G cap (m7G-cap) and a uracil at position three. We also demonstrate how PUCH interacts with PETISCO, a complex that binds to piRNA precursors4, and that this interaction enhances piRNA production in vivo. The identification of PUCH concludes the search for the 5'-end piRNA biogenesis factor in C. elegans and uncovers a type of RNA endonuclease formed by three SLFL proteins. Mammalian Schlafen (SLFN) genes have been associated with immunity5, exposing a molecular link between immune responses in mammals and deeply conserved RNA-based mechanisms that control transposable elements.

Correction: A modified TurboID approach identifies tissue-specific centriolar components in C. elegans.

[This corrects the article DOI: 10.1371/journal.pgen.1010150.].

Error detection and filtering of incompressible flow simulations for aeroacoustic predictions of human voice.

The presented filtering technique is proposed to detect errors and correct outliers inside the acoustic sources, respectively, the first time derivative of the incompressible pressure obtained from large eddy simulations with prescribed vocal fold motion using overlay mesh methods. Regarding the perturbed convective wave equation, the time derivative of the incompressible pressure is the primary sound source in the human phonation process. However, the incompressible pressure can be erroneous and have outliers when fulfilling the divergence-free constraint of the velocity field. This error is primarily occurring for non-conserving prescribed vocal fold motions. Therefore, the method based on a continuous stationary random process was designed to detect rare events in the time derivative of the pressure. The detected events are then localized and treated by a defined window function to increase their probability. As a consequence, the data quality of the non-linearly filtered data is enhanced significantly. Furthermore, the proposed method can also be used to assess convergence of the aeroacoustic source terms, and detect regions and time intervals, which show a non-converging behavior by an impulse-like structure.

Evaluation of Respiratory Particle Emission during Otorhinolaryngological Procedures in the Context of the SARS-CoV-2 Pandemic.

Understanding the risk of infection by routine medical examination is important for the protection of the medical personnel. In this study we investigated respiratory particles emitted by patients during routine otolaryngologic procedures and assessed the risks for the performing physician. We developed two experimental setups to measure aerosol and droplet emission during rigid/flexible laryngoscopy, rhinoscopy, pharyngoscopy, otoscopy, sonography and patient interview for subjects with and without masks. A high-speed-camera setup was used to detect ballistic droplets (approx. > 100 µm) and an aerosol-particle-sizer was used to detect aerosol particles in the range of 0.3 µm to 10 µm. Aerosol particle counts were highly increased for coughing and slightly increased for heavy breathing in subjects without masks. The highest aerosol particle counts occurred during rigid laryngoscopy. During laryngoscopy and rhinoscopy, the examiner was exposed to increased particle emission due to close proximity to the patient's face and provoked events such as coughing. However, even during sonography or otoscopy without a mask, aerosol particles were expelled close to the examiner. The physician's exposure to respiratory particles can be reduced by deliberate choice of examination technique depending on medical indication and the use of appropriate equipment for the examiners and the patients (e.g., FFP2 masks for both).

A modified TurboID approach identifies tissue-specific centriolar components in C. elegans.

Proximity-dependent labeling approaches such as BioID have been a great boon to studies of protein-protein interactions in the context of cytoskeletal structures such as centrosomes which are poorly amenable to traditional biochemical approaches like immunoprecipitation and tandem affinity purification. Yet, these methods have so far not been applied extensively to invertebrate experimental models such as C. elegans given the long labeling times required for the original promiscuous biotin ligase variant BirA*. Here, we show that the recently developed variant TurboID successfully probes the interactomes of both stably associated (SPD-5) and dynamically localized (PLK-1) centrosomal components. We further develop an indirect proximity labeling method employing a GFP nanobody-TurboID fusion, which allows the identification of protein interactors in a tissue-specific manner in the context of the whole animal. Critically, this approach utilizes available endogenous GFP fusions, avoiding the need to generate multiple additional strains for each target protein and the potential complications associated with overexpressing the protein from transgenes. Using this method, we identify homologs of two highly conserved centriolar components, Cep97 and BLD10/Cep135, which are present in various somatic tissues of the worm. Surprisingly, neither protein is expressed in early embryos, likely explaining why these proteins have escaped attention until now. Our work expands the experimental repertoire for C. elegans and opens the door for further studies of tissue-specific variation in centrosome architecture.

Release of CHK-2 from PPM-1.D anchorage schedules meiotic entry.

Transition from the stem/progenitor cell fate to meiosis is mediated by several redundant posttranscriptional regulatory pathways in Caenorhabditis elegans. Interfering with all three branches causes tumorous germ lines. SCFPROM-1 comprises one branch and mediates a scheduled degradation step at entry into meiosis. prom-1 mutants show defects in the timely initiation of meiotic prophase I events, resulting in high rates of embryonic lethality. Here, we identify the phosphatase PPM-1.D/Wip1 as crucial substrate for PROM-1. We report that PPM-1.D antagonizes CHK-2 kinase, a key regulator for meiotic prophase initiation, including DNA double-strand breaks, chromosome pairing, and synaptonemal complex formation. We propose that PPM-1.D controls the amount of active CHK-2 via both catalytic and noncatalytic activities; notably, noncatalytic regulation seems to be crucial at meiotic entry. PPM-1.D sequesters CHK-2 at the nuclear periphery, and programmed SCFPROM-1-mediated degradation of PPM-1.D liberates the kinase and promotes meiotic entry.

Structural basis of PETISCO complex assembly during piRNA biogenesis in C. elegans.

Piwi-interacting RNAs (piRNAs) constitute a class of small RNAs that bind PIWI proteins and are essential to repress transposable elements in the animal germline, thereby promoting genome stability and maintaining fertility. C. elegans piRNAs (21U RNAs) are transcribed individually from minigenes as precursors that require 5' and 3' processing. This process depends on the PETISCO complex, consisting of four proteins: IFE-3, TOFU-6, PID-3, and ERH-2. We used biochemical and structural biology approaches to characterize the PETISCO architecture and its interaction with RNA, together with its effector proteins TOST-1 and PID-1. These two proteins define different PETISCO functions: PID-1 governs 21U processing, whereas TOST-1 links PETISCO to an unknown process essential for early embryogenesis. Here, we show that PETISCO forms an octameric assembly with each subunit present in two copies. Determination of structures of the TOFU-6/PID-3 and PID-3/ERH-2 subcomplexes, supported by in vivo studies of subunit interaction mutants, allows us to propose a model for the formation of the TOFU-6/PID-3/ERH-2 core complex and its functionality in germ cells and early embryos. Using NMR spectroscopy, we demonstrate that TOST-1 and PID-1 bind to a common surface on ERH-2, located opposite its PID-3 binding site, explaining how PETISCO can mediate different cellular roles.

A ribonuclease III involved in virulence of Mucorales fungi has evolved to cut exclusively single-stranded RNA.

Members of the ribonuclease III (RNase III) family regulate gene expression by processing double-stranded RNA (dsRNA). This family includes eukaryotic Dicer and Drosha enzymes that generate small dsRNAs in the RNA interference (RNAi) pathway. The fungus Mucor lusitanicus, which causes the deadly infection mucormycosis, has a complex RNAi system encompassing a non-canonical RNAi pathway (NCRIP) that regulates virulence by degrading specific mRNAs. In this pathway, Dicer function is replaced by R3B2, an atypical class I RNase III, and small single-stranded RNAs (ssRNAs) are produced instead of small dsRNA as Dicer-dependent RNAi pathways. Here, we show that R3B2 forms a homodimer that binds to ssRNA and dsRNA molecules, but exclusively cuts ssRNA, in contrast to all known RNase III. The dsRNA cleavage inability stems from its unusual RNase III domain (RIIID) because its replacement by a canonical RIIID allows dsRNA processing. A crystal structure of R3B2 RIIID resembles canonical RIIIDs, despite the low sequence conservation. However, the groove that accommodates dsRNA in canonical RNases III is narrower in the R3B2 homodimer, suggesting that this feature could be responsible for the cleavage specificity for ssRNA. Conservation of this activity in R3B2 proteins from other mucormycosis-causing Mucorales fungi indicates an early evolutionary acquisition.

3D-FV-FE Aeroacoustic Larynx Model for Investigation of Functional Based Voice Disorders.

For the clinical analysis of underlying mechanisms of voice disorders, we developed a numerical aeroacoustic larynx model, called simVoice, that mimics commonly observed functional laryngeal disorders as glottal insufficiency and vibrational left-right asymmetries. The model is a combination of the Finite Volume (FV) CFD solver Star-CCM+ and the Finite Element (FE) aeroacoustic solver CFS++. simVoice models turbulence using Large Eddy Simulations (LES) and the acoustic wave propagation with the perturbed convective wave equation (PCWE). Its geometry corresponds to a simplified larynx and a vocal tract model representing the vowel /a/. The oscillations of the vocal folds are externally driven. In total, 10 configurations with different degrees of functional-based disorders were simulated and analyzed. The energy transfer between the glottal airflow and the vocal folds decreases with an increasing glottal insufficiency and potentially reflects the higher effort during speech for patients being concerned. This loss of energy transfer may also have an essential influence on the quality of the sound signal as expressed by decreasing sound pressure level (SPL), Cepstral Peak Prominence (CPP), and Vocal Efficiency (VE). Asymmetry in the vocal fold oscillations also reduces the quality of the sound signal. However, simVoice confirmed previous clinical and experimental observations that a high level of glottal insufficiency worsens the acoustic signal quality more than oscillatory left-right asymmetry. Both symptoms in combination will further reduce the quality of the sound signal. In summary, simVoice allows for detailed analysis of the origins of disordered voice production and hence fosters the further understanding of laryngeal physiology, including occurring dependencies. A current walltime of 10 h/cycle is, with a prospective increase in computing power, auspicious for a future clinical use of simVoice.

Hybrid aeroacoustic approach for the efficient numerical simulation of human phonation.

A hybrid aeroacoustic approach was developed for the efficient numerical computation of human phonation. In the first step, an incompressible flow simulation on a three-dimensional (3 D) computational grid, which is capable of resolving all relevant turbulent scales, is performed using STARCCM+ and finite volume method. In the second step, the acoustic source terms on the flow grid are computed and a conservative interpolation to the acoustic grid is performed. Finally, the perturbed convective wave equation is solved to obtain the acoustic field in 3 D with the finite element solver CFS++. Thereby, the conservative transformation of the acoustic sources from the flow grid to the acoustic grid is a key step to allow coarse acoustic grids without reducing accuracy. For this transformation, two different interpolation strategies are compared and grid convergence is assessed. Overall, 16 simulation setups are compared. The initial (267 000 degrees of freedom) and the optimized (21 265 degrees of freedom) simulation setup were validated by measurements of a synthetic larynx model. To conclude, the total computational time of the acoustic simulation is reduced by 95% compared to the initial simulation setup without a significant reduction of accuracy, being 7%, in the frequency range of interest.

Purification and Reconstitution of the S. cerevisiae TRAMP and Ski Complexes for Biochemical and Structural Studies.

The RNA exosome is a macromolecular machine that degrades a large variety of RNAs from their 3'-end. It comprises the major 3'-to-5' exonuclease in the cell, completely degrades erroneous and overly abundant RNAs, and is also involved in the precise processing of RNAs. To degrade transcripts both specifically and efficiently the exosome functions together with compartment-specific cofactors. In the yeast S. cerevisiae, the exosome associates with the Ski complex in the cytoplasm and with Mtr4 alone or with Mtr4 as part of the TRAMP complex in the nucleus. Here we describe how to produce, purify, and assemble the Ski and TRAMP complexes from S. cerevisiae.

The MTR4 helicase recruits nuclear adaptors of the human RNA exosome using distinct arch-interacting motifs.

The nuclear exosome and its essential co-factor, the RNA helicase MTR4, play crucial roles in several RNA degradation pathways. Besides unwinding RNA substrates for exosome-mediated degradation, MTR4 associates with RNA-binding proteins that function as adaptors in different RNA processing and decay pathways. Here, we identify and characterize the interactions of human MTR4 with a ribosome processing adaptor, NVL, and with ZCCHC8, an adaptor involved in the decay of small nuclear RNAs. We show that the unstructured regions of NVL and ZCCHC8 contain short linear motifs that bind the MTR4 arch domain in a mutually exclusive manner. These short sequences diverged from the arch-interacting motif (AIM) of yeast rRNA processing factors. Our results suggest that nuclear exosome adaptors have evolved canonical and non-canonical AIM sequences to target human MTR4 and demonstrate the versatility and specificity with which the MTR4 arch domain can recruit a repertoire of different RNA-binding proteins.

To Process or to Decay: A Mechanistic View of the Nuclear RNA Exosome.

The RNA exosome was originally discovered in yeast as an RNA-processing complex required for the maturation of 5.8S ribosomal RNA (rRNA), one of the constituents of the large ribosomal subunit. The exosome is now known in eukaryotes as the major 3'-5' RNA degradation machine involved in numerous processing, turnover, and surveillance pathways, both in the nucleus and the cytoplasm. Yet its role in maturing the 5.8S rRNA in the pre-60S ribosomal particle remains probably the most intricate and emblematic among its functions, as it involves all the RNA unwinding, degradation, and trimming activities embedded in this macromolecular complex. Here, we propose a comprehensive mechanistic model, based on current biochemical and structural data, explaining the dual functions of the nuclear exosome-the constructive versus the destructive mode.

Distinct and evolutionary conserved structural features of the human nuclear exosome complex.

The nuclear RNA exosome complex mediates the processing of structured RNAs and the decay of aberrant non-coding RNAs, an important function particularly in human cells. Most mechanistic studies to date have focused on the yeast system. Here, we reconstituted and studied the properties of a recombinant 14-subunit human nuclear exosome complex. In biochemical assays, the human exosome embeds a longer RNA channel than its yeast counterpart. The 3.8 Å resolution cryo-EM structure of the core complex bound to a single-stranded RNA reveals that the RNA channel path is formed by two distinct features of the hDIS3 exoribonuclease: an open conformation and a domain organization more similar to bacterial RNase II than to yeast Rrp44. The cryo-EM structure of the holo-complex shows how obligate nuclear cofactors position the hMTR4 helicase at the entrance of the core complex, suggesting a striking structural conservation from lower to higher eukaryotes.

Structure of the nuclear exosome captured on a maturing preribosome.

The RNA exosome complex processes and degrades a wide range of transcripts, including ribosomal RNAs (rRNAs). We used cryo-electron microscopy to visualize the yeast nuclear exosome holocomplex captured on a precursor large ribosomal subunit (pre-60S) during 7S-to-5.8S rRNA processing. The cofactors of the nuclear exosome are sandwiched between the ribonuclease core complex (Exo-10) and the remodeled "foot" structure of the pre-60S particle, which harbors the 5.8S rRNA precursor. The exosome-associated helicase Mtr4 recognizes the preribosomal substrate by docking to specific sites on the 25S rRNA, captures the 3' extension of the 5.8S rRNA, and channels it toward Exo-10. The structure elucidates how the exosome forms a structural and functional unit together with its massive pre-60S substrate to process rRNA during ribosome maturation.

Reconstitution of the complete pathway of ITS2 processing at the pre-ribosome.

Removal of internal transcribed spacer 2 (ITS2) from pre-ribosomal RNA is essential to make functional ribosomes. This complicated processing reaction begins with a single endonucleolytic cleavage followed by exonucleolytic trimming at both new cleavage sites to generate mature 5.8S and 25S rRNA. We reconstituted the 7S→5.8S processing branch within ITS2 using purified exosome and its nuclear cofactors. We find that both Rrp44's ribonuclease activities are required for initial RNA shortening followed by hand over to the exonuclease Rrp6. During the in vitro reaction, ITS2-associated factors dissociate and the underlying 'foot' structure of the pre-60S particle is dismantled. 7S pre-rRNA processing is independent of 5S RNP rotation, but 26S→25S trimming is a precondition for subsequent 7S→5.8S processing. To complete the in vitro assay, we reconstituted the entire cycle of ITS2 removal with a total of 18 purified factors, catalysed by the integrated activities of the two participating RNA-processing machines, the Las1 complex and nuclear exosome.

Structural insights into the interaction of the nuclear exosome helicase Mtr4 with the preribosomal protein Nop53.

The nuclear exosome and the associated RNA helicase Mtr4 participate in the processing of several ribonucleoprotein particles (RNP), including the maturation of the large ribosomal subunit (60S). S. cerevisiae Mtr4 interacts directly with Nop53, a ribosomal biogenesis factor present in late pre-60S particles containing precursors of the 5.8S rRNA. The Mtr4-Nop53 interaction plays a pivotal role in the maturation of the 5.8S rRNA, providing a physical link between the nuclear exosome and the pre-60S RNP. An analogous interaction between Mtr4 and another ribosome biogenesis factor, Utp18, directs the exosome to an earlier preribosomal particle. Nop53 and Utp18 contain a similar Mtr4-binding motif known as the arch-interacting motif (AIM). Here, we report the 3.2 Å resolution crystal structure of S. cerevisiae Mtr4 bound to the interacting region of Nop53, revealing how the KOW domain of the helicase recognizes the AIM sequence of Nop53 with a network of hydrophobic and electrostatic interactions. The AIM-interacting residues are conserved in Mtr4 and are not present in the related cytoplasmic helicase Ski2, rationalizing the specificity and versatility of Mtr4 in the recognition of different AIM-containing proteins. Using nuclear magnetic resonance (NMR), we show that the KOW domain of Mtr4 can simultaneously bind an AIM-containing protein and a structured RNA at adjacent surfaces, suggesting how it can dock onto RNPs. The KOW domains of exosome-associated helicases thus appear to have evolved from the KOW domains of ribosomal proteins and to function as RNP-binding modules in the context of the nuclear exosome.

Mpp6 Incorporation in the Nuclear Exosome Contributes to RNA Channeling through the Mtr4 Helicase.

The RNA-degrading exosome mediates the processing and decay of many cellular transcripts. In the yeast nucleus, the ubiquitous 10-subunit exosome core complex (Exo-9-Rrp44) functions with four conserved cofactors (Rrp6, Rrp47, Mtr4, and Mpp6). Biochemical and structural studies to date have shed insights into the mechanisms of the exosome core and its nuclear cofactors, with the exception of Mpp6. We report the 3.2-Å resolution crystal structure of a S. cerevisiae Exo-9-Mpp6 complex, revealing how linear motifs in the Mpp6 middle domain bind Rrp40 via evolutionary conserved residues. In particular, Mpp6 binds near a tryptophan residue of Rrp40 that is mutated in human patients suffering from pontocerebellar hypoplasia. Using biochemical assays, we show that Mpp6 is required for the ability of Mtr4 to extend the trajectory of an RNA entering the exosome core, suggesting that it promotes the channeling of substrates from the nuclear helicase to the processive RNase.

Structure of the RBM7-ZCCHC8 core of the NEXT complex reveals connections to splicing factors.

The eukaryotic RNA exosome participates extensively in RNA processing and degradation. In human cells, three accessory factors (RBM7, ZCCHC8 and hMTR4) interact to form the nuclear exosome targeting (NEXT) complex, which directs a subset of non-coding RNAs for exosomal degradation. Here we elucidate how RBM7 is incorporated in the NEXT complex. We identify a proline-rich segment of ZCCHC8 as the interaction site for the RNA-recognition motif (RRM) of RBM7 and present the crystal structure of the corresponding complex at 2.0 Å resolution. On the basis of the structure, we identify a proline-rich segment within the splicing factor SAP145 with strong similarity to ZCCHC8. We show that this segment of SAP145 not only binds the RRM region of another splicing factor SAP49 but also the RRM of RBM7. These dual interactions of RBM7 with the exosome and the spliceosome suggest a model whereby NEXT might recruit the exosome to degrade intronic RNAs.

The conformational plasticity of eukaryotic RNA-dependent ATPases.

RNA helicases are present in all domains of life and participate in almost all aspects of RNA metabolism, from transcription and processing to translation and decay. The diversity of pathways and substrates that they act on is reflected in the diversity of their individual functions, structures, and mechanisms. However, RNA helicases also share hallmark properties. At the functional level, they promote rearrangements of RNAs and RNP particles by coupling nucleic acid binding and release with ATP hydrolysis. At the molecular level, they contain two domains homologous to the bacterial RecA recombination protein. This conserved catalytic core is flanked by additional domains, which typically regulate the ATPase activity in cis. Binding to effector proteins targets or regulates the ATPase activity in trans. Structural and biochemical studies have converged on the plasticity of RNA helicases as a fundamental property that is used to control their timely activation in the cell. In this review, we focus on the conformational regulation of conserved eukaryotic RNA helicases.