Noncanonical processing by animal Microprocessor.

Microprocessor (MP), DROSHA-DGCR8, processes primary miRNA transcripts (pri-miRNAs) to initiate miRNA biogenesis. The canonical cleavage mechanism of MP has been extensively investigated and comprehensively validated for two decades. However, this canonical mechanism cannot account for the processing of certain pri-miRNAs in animals. In this study, by conducting high-throughput pri-miRNA cleavage assays for approximately 260,000 pri-miRNA sequences, we discovered and comprehensively characterized a noncanonical cleavage mechanism of MP. This noncanonical mechanism does not need several RNA and protein elements essential for the canonical mechanism; instead, it utilizes previously unrecognized DROSHA dsRNA recognition sites (DRESs). Interestingly, the noncanonical mechanism is conserved across animals and plays a particularly significant role in C. elegans. Our established noncanonical mechanism elucidates MP cleavage in numerous RNA substrates unaccounted for by the canonical mechanism in animals. This study suggests a broader substrate repertoire of animal MPs and an expanded regulatory landscape for miRNA biogenesis.

A quantitative map of human primary microRNA processing sites.

Maturation of canonical microRNA (miRNA) is initiated by DROSHA that cleaves the primary transcript (pri-miRNA). More than 1,800 miRNA loci are annotated in humans, but it remains largely unknown whether and at which sites pri-miRNAs are cleaved by DROSHA. Here, we performed in vitro processing on a full set of human pri-miRNAs (miRBase version 21) followed by sequencing. This comprehensive profiling enabled us to classify miRNAs on the basis of DROSHA dependence and map their cleavage sites with respective processing efficiency measures. Only 758 pri-miRNAs are confidently processed by DROSHA, while the majority may be non-canonical or false entries. Analyses of the DROSHA-dependent pri-miRNAs show key cis-elements for processing. We observe widespread alternative processing and unproductive cleavage events such as "nick" or "inverse" processing. SRSF3 is a broad-acting auxiliary factor modulating alternative processing and suppressing unproductive processing. The profiling data and methods developed in this study will allow systematic analyses of miRNA regulation.

Genomic Clustering Facilitates Nuclear Processing of Suboptimal Pri-miRNA Loci.

Nuclear processing of most miRNAs is mediated by Microprocessor, comprised of RNase III enzyme Drosha and its cofactor DGCR8. Here, we uncover a hidden layer of Microprocessor regulation via studies of Dicer-independent mir-451, which is clustered with canonical mir-144. Although mir-451 is fully dependent on Drosha/DGCR8, its short stem and small terminal loop render it an intrinsically weak Microprocessor substrate. Thus, it must reside within a cluster for normal biogenesis, although the identity and orientation of its neighbor are flexible. We use DGCR8 tethering assays and operon structure-function assays to demonstrate that local recruitment and transfer of Microprocessor enhances suboptimal substrate processing. This principle applies more broadly since genomic analysis indicates suboptimal canonical miRNAs are enriched in operons, and we validate several of these experimentally. Proximity-based enhancement of suboptimal hairpin processing provides a rationale for genomic retention of certain miRNA operons and may explain preferential evolutionary emergence of miRNA operons.

Structural Basis for pri-miRNA Recognition by Drosha.

A commencing and critical step in miRNA biogenesis involves processing of pri-miRNAs in the nucleus by Microprocessor. An important, but not completely understood, question is how Drosha, the catalytic subunit of Microprocessor, binds pri-miRNAs and correctly specifies cleavage sites. Here we report the cryoelectron microscopy structures of the Drosha-DGCR8 complex with and without a pri-miRNA. The RNA-bound structure provides direct visualization of the tertiary structure of pri-miRNA and shows that a helix hairpin in the extended PAZ domain and the mobile basic (MB) helix in the RNase IIIa domain of Drosha coordinate to recognize the single-stranded to double-stranded junction of RNA, whereas the dsRNA binding domain makes extensive contacts with the RNA stem. Furthermore, the RNA-free structure reveals an autoinhibitory conformation of the PAZ helix hairpin. These findings provide mechanistic insights into pri-miRNA cleavage site selection and conformational dynamics governing pri-miRNA recognition by the catalytic component of Microprocessor.

Functional Atlas of Primary miRNA Maturation by the Microprocessor.

Primary microRNAs (miRNAs) are the precursors of miRNAs that modulate the expression of most mRNAs in humans. They fold up into a hairpin structure that is cleaved at its base by an enzyme complex known as the Microprocessor (Drosha/DGCR8). While many of the molecular details are known, a complete understanding of what features distinguish primary miRNA from hairpin structures in other transcripts is still lacking. We develop a massively parallel functional assay termed Dro-seq (Drosha sequencing) that enables testing of hundreds of known primary miRNA substrates and thousands of single-nucleotide variants. We find an additional feature of primary miRNAs, called Shannon entropy, describing the structural ensemble important for processing. In a deep mutagenesis experiment, we observe particular apical loop U bases, likely recognized by DGCR8, are important for efficient processing. These findings build on existing knowledge about primary miRNA maturation by the Microprocessor and further explore the substrate RNA sequence-structure relationship.

Cryo-EM Structures of Human Drosha and DGCR8 in Complex with Primary MicroRNA.

Metazoan microRNAs require specific maturation steps initiated by Microprocessor, comprising Drosha and DGCR8. Lack of structural information for the assembled complex has hindered an understanding of how Microprocessor recognizes primary microRNA transcripts (pri-miRNAs). Here we present a cryoelectron microscopy structure of human Microprocessor with a pri-miRNA docked in the active site, poised for cleavage. The basal junction is recognized by a four-way intramolecular junction in Drosha, triggered by the Belt and Wedge regions that clamp over the ssRNA. The belt is important for efficiency and accuracy of pri-miRNA processing. Two dsRBDs form a molecular ruler to measure the stem length between the two dsRNA-ssRNA junctions. The specific organization of the dsRBDs near the apical junction is independent of Drosha core domains, as observed in a second structure in the partially docked state. Collectively, we derive a molecular model to explain how Microprocessor recognizes a pri-miRNA and accurately identifies the cleavage site.

SAFB2 Enables the Processing of Suboptimal Stem-Loop Structures in Clustered Primary miRNA Transcripts.

Many microRNAs (miRNAs) are generated from primary transcripts containing multiple clustered stem-loop structures that are thought to be recognized and cleaved by the Microprocessor complex as independent units. Here, we uncover an unexpected mode of processing of the bicistronic miR-15a-16-1 cluster. We find that the primary miR-15a stem-loop is not processed on its own but that the presence of the neighboring primary miR-16-1 stem-loop on the same transcript can compensate for this deficiency in cis. Using a CRISPR/Cas9 screen, we identify SAFB2 (scaffold attachment factor B2) as an essential co-factor in this miR-16-1-assisted pri-miR-15 cleavage and describe SAFB2 as an accessory protein of the Microprocessor. Notably, SAFB2-mediated cleavage expands to other clustered pri-miRNAs, indicating a general mechanism. Together, our study reveals an unrecognized function of SAFB2 in miRNA processing and suggests a scenario in which SAFB2 enables the binding and processing of suboptimal Microprocessor substrates in clustered primary miRNA transcripts.

Oncogenic Biogenesis of pri-miR-17∼92 Reveals Hierarchy and Competition among Polycistronic MicroRNAs.

The microRNAs encoded by the miR-17∼92 polycistron are commonly overexpressed in cancer and orchestrate a wide range of oncogenic functions. Here, we identify a mechanism for miR-17∼92 oncogenic function through the disruption of endogenous microRNA (miRNA) processing. We show that, upon oncogenic overexpression of the miR-17∼92 primary transcript (pri-miR-17∼92), the microprocessor complex remains associated with partially processed intermediates that aberrantly accumulate. These intermediates reflect a series of hierarchical and conserved steps in the early processing of the pri-miR-17∼92 transcript. Encumbrance of the microprocessor by miR-17∼92 intermediates leads to the broad but selective downregulation of co-expressed polycistronic miRNAs, including miRNAs derived from tumor-suppressive miR-34b/c and from the Dlk1-Dio3 polycistrons. We propose that the identified steps of polycistronic miR-17∼92 biogenesis contribute to the oncogenic re-wiring of gene regulation networks. Our results reveal previously unappreciated functional paradigms for polycistronic miRNAs in cancer.

Parameters of clustered suboptimal miRNA biogenesis.

The nuclear cleavage of a suboptimal primary miRNA hairpin by the Drosha/DGCR8 complex ("Microprocessor") can be enhanced by an optimal miRNA neighbor, a phenomenon termed cluster assistance. Several features and biological impacts of this new layer of miRNA regulation are not fully known. Here, we elucidate the parameters of cluster assistance of a suboptimal miRNA and also reveal competitive interactions amongst optimal miRNAs within a cluster. We exploit cluster assistance as a functional assay for suboptimal processing and use this to invalidate putative suboptimal substrates, as well as identify a "solo" suboptimal miRNA. Finally, we report complexity in how specific mutations might affect the biogenesis of clustered miRNAs in disease contexts. This includes how an operon context can buffer the effect of a deleterious processing variant, but reciprocally how a point mutation can have a nonautonomous effect to impair the biogenesis of a clustered, suboptimal, neighbor. These data expand our knowledge regarding regulated miRNA biogenesis in humans and represent a functional assay for empirical definition of suboptimal Microprocessor substrates.

The plant FYVE domain-containing protein FREE1 associates with microprocessor components to repress miRNA biogenesis.

FYVE domain protein required for endosomal sorting 1 (FREE1), originally identified as a plant-specific component of the endosomal sorting complex required for transport (ESCRT) machinery, plays diverse roles either in endosomal sorting in the cytoplasm or in transcriptional regulation of abscisic acid signaling in the nucleus. However, to date, a role for FREE1 or other ESCRT components in the regulation of plant miRNA biology has not been discovered. Here, we demonstrate a nuclear function of FREE1 as a cofactor in miRNA biogenesis in plants. FREE1 directly interacts with the plant core microprocessor component CPL1 in nuclear bodies and disturbs the association between HYL1, SE and CPL1. Inactivation of FREE1 in the nucleus increases the binding affinity between HYL1, SE, and CPL1 and causes a transition of HYL1 from the inactive hyperphosphorylated version to the active hypophosphorylated form, thereby promoting miRNA biogenesis. Our results suggest that FREE1 has evolved as a negative regulator of miRNA biogenesis and provides evidence for a link between FYVE domain-containing proteins and miRNA biogenesis in plants.

Genome-wide Mapping of DROSHA Cleavage Sites on Primary MicroRNAs and Noncanonical Substrates.

MicroRNA (miRNA) maturation is initiated by DROSHA, a double-stranded RNA (dsRNA)-specific RNase III enzyme. By cleaving primary miRNAs (pri-miRNAs) at specific positions, DROSHA serves as a main determinant of miRNA sequences and a highly selective gatekeeper for the canonical miRNA pathway. However, the sites of DROSHA-mediated processing have not been annotated, and it remains unclear to what extent DROSHA functions outside the miRNA pathway. Here, we establish a protocol termed "formaldehyde crosslinking, immunoprecipitation, and sequencing (fCLIP-seq)," which allows identification of DROSHA cleavage sites at single-nucleotide resolution. fCLIP identifies numerous processing sites, suggesting widespread end modifications during miRNA maturation. fCLIP also finds many pri-miRNAs that undergo alternative processing, yielding multiple miRNA isoforms. Moreover, we discovered dozens of DROSHA substrates on non-miRNA loci, which may serve as cis-elements for DROSHA-mediated gene regulation. We anticipate that fCLIP-seq could be a general tool for investigating interactions between dsRNA-binding proteins and structured RNAs.

A Spectroscopic Reflectance-Based Low-Cost Thickness Measurement System for Thin Films: Development and Testing.

The requirement for alternatives in roll-to-roll (R2R) processing to expand thin film inspection in wider substrates at lower costs and reduced dimensions, and the need to enable newer control feedback options for these types of processes, represents an opportunity to explore the applicability of newer reduced-size spectrometers sensors. This paper presents the hardware and software development of a novel low-cost spectroscopic reflectance system using two state-of-the-art sensors for thin film thickness measurements. The parameters to enable the thin film measurements using the proposed system are the light intensity for two LEDs, the microprocessor integration time for both sensors and the distance from the thin film standard to the device light channel slit for reflectance calculations. The proposed system can deliver better-fit errors compared with a HAL/DEUT light source using two methods: curve fitting and interference interval. By enabling the curve fitting method, the lowest root mean squared error (RMSE) obtained for the best combination of components was 0.022 and the lowest normalised mean squared error (MSE) was 0.054. The interference interval method showed an error of 0.09 when comparing the measured with the expected modelled value. The proof of concept in this research work enables the expansion of multi-sensor arrays for thin film thickness measurements and the potential application in moving environments.

A Measurement System for the Environmental Load Assessment of a Scale Ship Model-Part II.

In the process of ship motion control system design, it is necessary to take into account the impact of environmental disturbances such as winds, waves and sea currents. The commonly used representatives of wave influences in this area are the unidirectional wave power spectral density functions describing sea waves of different form: long-crested, fully developed waves, developing wind waves or multi-modal waves (e.g., with swell). The existing standard PSD models describe the surge of open sea or ocean. However, they are inadequate in the case of control system testing of scale ship models for sailing in open water areas such as lakes or test pools. This paper presents a study of wind-generated wave PSD estimations for a small lake used as a test area for free-running scale ships. The publication provides a brief overview of the wave spectral density functions commonly used for control system design. A measurement instrument using the idea of a water-induced variable capacitance that works synchronously with the wind sensors is also described. The process of collected data analysis is presented. As a result of the study, a series of empirical spectral density functions of lake waves for different wind speeds are obtained. They correspond to the rescaled, two-parametric ITTC model.

Advanced System-on-Chip Field-Programmable-Gate-Array-Powered Data Acquisition System for Pixel Detectors.

Particle detector systems require data acquisition systems (DAQs) as their back-end. This paper presents a new edge-computing DAQ that is capable of handling multiple pixel detectors simultaneously and was designed for particle-tracking experiments. The system was designed for the ROC4SENS readout chip, but its control logic can be adapted for other pixel detectors. The DAQ was based on a system-on-chip FPGA (SoC FPGA), which includes an embedded microprocessor running a fully functional Linux system. An application using a client-server architecture was developed to facilitate remote control and data visualization. The comprehensive DAQ is very compact, thus reducing the typical hardware load in particle tracking experiments, especially during the obligatory characterization of particle telescopes.

CtIP suppresses primary microRNA maturation and promotes metastasis of colon cancer cells in a xenograft mouse model.

miRNAs are important regulators of eukaryotic gene expression. The post-transcriptional maturation of miRNAs is controlled by the Drosha-DiGeorge syndrome critical region gene 8 (DGCR8) microprocessor. Dysregulation of miRNA biogenesis has been implicated in the pathogenesis of human diseases, including cancers. C-terminal-binding protein-interacting protein (CtIP) is a well-known DNA repair factor that promotes the processing of DNA double-strand break (DSB) to initiate homologous recombination-mediated DSB repair. However, it was unclear whether CtIP has other unknown cellular functions. Here, we aimed to uncover the roles of CtIP in miRNA maturation and cancer cell metastasis. We found that CtIP is a potential regulatory factor that suppresses the processing of miRNA primary transcripts (pri-miRNA). CtIP directly bound to both DGCR8 and pri-miRNAs through a conserved Sae2-like domain, reduced the binding of Drosha to DGCR8 and pri-miRNA substrate, and inhibited processing activity of Drosha complex. CtIP depletion significantly increased the expression levels of a subset of mature miRNAs, including miR-302 family members that are associated with tumor progression and metastasis in several cancer types. We also found that CtIP-inhibited miRNAs, such as miR-302 family members, are not crucial for DSB repair. However, increase of miR-302b levels or loss of CtIP function severely suppressed human colon cancer cell line tumor cell metastasis in a mouse xenograft model. These studies reveal a previously unrecognized mechanism of CtIP in miRNA processing and tumor metastasis that represents a new function of CtIP in cancer.

Biogenesis, conservation, and function of miRNA in liverworts.

MicroRNAs (miRNAs) are small non-coding endogenous RNA molecules, 18-24 nucleotides long, that control multiple gene regulatory pathways via post-transcriptional gene silencing in eukaryotes. To develop a comprehensive picture of the evolutionary history of miRNA biogenesis and action in land plants, studies on bryophyte representatives are needed. Here, we review current understanding of liverwort MIR gene structure, miRNA biogenesis, and function, focusing on the simple thalloid Pellia endiviifolia and the complex thalloid Marchantia polymorpha. We review what is known about conserved and non-conserved miRNAs, their targets, and the functional implications of miRNA action in M. polymorpha and P. endiviifolia. We note that most M. polymorpha miRNAs are encoded within protein-coding genes and provide data for 23 MIR gene structures recognized as independent transcriptional units. We identify M. polymorpha genes involved in miRNA biogenesis that are homologous to those identified in higher plants, including those encoding core microprocessor components and other auxiliary and regulatory proteins that influence the stability, folding, and processing of pri-miRNAs. We analyzed miRNA biogenesis proteins and found similar domain architecture in most cases. Our data support the hypothesis that almost all miRNA biogenesis factors in higher plants are also present in liverworts, suggesting that they emerged early during land plant evolution.

Benefits of a microprocessor-controlled prosthetic foot for ascending and descending slopes.

BACKGROUND: Prosthetic feet are prescribed for persons with a lower-limb amputation to restore lost mobility. However, due to limited adaptability of their ankles and springs, situations like walking on slopes or uneven ground remain challenging. This study investigated to what extent a microprocessor-controlled prosthetic foot (MPF) facilitates walking on slopes. METHODS: Seven persons each with a unilateral transtibial amputation (TTA) and unilateral transfemoral amputation (TFA) as well as ten able-bodied subjects participated. Participants were studied while using a MPF and their prescribed standard feet with fixed ankle attachments. The study investigated ascending and descending a 10° slope. Kinematic and kinetic data were recorded with a motion capture system. Biomechanical parameters, in particular leg joint angles, shank orientation and external joint moments of the prosthetics side were calculated. RESULTS: Prosthetic feet- and subject group-dependent joint angle and moment characteristics were observed for both situations. The MPF showed a larger and situation-dependent ankle range of motion compared to the standard feet. Furthermore, it remained in a dorsiflexed position during swing. While ascending, the MPF adapted the dorsiflexion moment and reduced the knee extension moment. At vertical shank orientation, it reduced the knee extension moment by 26% for TFA and 49% for TTA compared to the standard feet. For descending, differences between feet in the biomechanical knee characteristics were found for the TTA group, but not for the TFA group. At the vertical shank angle during slope descent, TTA demonstrated a behavior of the ankle moment similar to able-bodied controls when using the MPF. CONCLUSIONS: The studied MPF facilitated walking on slopes by adapting instantaneously to inclinations and, thus, easing the forward rotation of the leg over the prosthetic foot compared to standard feet with a fixed ankle attachment with amputation-level dependent effect sizes. It assumed a dorsiflexed ankle angle during swing, enabled a larger ankle range of motion and reduced the moments acting on the residual knee of TTA compared to the prescribed prosthetic standard feet. For individuals with TFA, the prosthetic knee joint seems to play a more crucial role for walking on ramps than the foot.

Development of an Artificial Neural Network Algorithm Embedded in an On-Site Sensor for Water Level Forecasting.

Extreme weather events cause stream overflow and lead to urban inundation. In this study, a decentralized flood monitoring system is proposed to provide water level predictions in streams three hours ahead. The customized sensor in the system measures the water levels and implements edge computing to produce future water levels. It is very different from traditional centralized monitoring systems and considered an innovation in the field. In edge computing, traditional physics-based algorithms are not computationally efficient if microprocessors are used in sensors. A correlation analysis was performed to identify key factors that influence the variations in the water level forecasts. For example, the second-order difference in the water level is considered to represent the acceleration or deacceleration of a water level rise. According to different input factors, three artificial neural network (ANN) models were developed. Four streams or canals were selected to test and evaluate the performance of the models. One case was used for model training and testing, and the others were used for model validation. The results demonstrated that the ANN model with the second-order water level difference as an input factor outperformed the other ANN models in terms of RMSE. The customized microprocessor-based sensor with an embedded ANN algorithm can be adopted to improve edge computing capabilities and support emergency response and decision making.

Measurement System for the Environmental Load Assessment of the Scale Ship Model.

The forces and moments acting on a marine vessel caused by the wind are most often modeled based on its speed measured at a standard 10 m above the sea level. There exist numerous well-known methods for modeling wind speed in such conditions. These models, by nature, are inadequate for simulating wind disturbances for free-running scale ship models sailing on lakes. Such scale models are being used increasingly for design and testing modern ship motion control systems. The paper describes the hardware and methodology used in measuring wind speed at low altitudes above the lake level. The system consists of two ultrasonic anemometers supplemented with wave sensor acting as a capacitor immersed partially in the water. Obtained measurement results show clear similarity to the values gathered during full-scale experiments. Analysis of the power spectral density functions of turbulence measured for different mean wind speeds over the lake, indicates that, at the present stage of research, the best model of wind turbulence at low altitude above the lake level can be obtained by assembling four of the known, standard turbulence models.

DGCR8 Microprocessor Subunit Mutation and Expression Deregulation in Thyroid Lesions.

DGCR8 emerged recently as miRNAs biogenesis pathway protein with a highlighted role in thyroid disease. This study aimed to characterize this miRNA biogenesis component, in particular the p.(E518K) mutation and DGCR8 expression in a series of thyroid lesions. The series of thyroid lesions was genotyped for the c.1552G>A p.(E518K) mutation. When frozen tissue was available, DGCR8 mRNA expression was analysed by qPCR. Formalin-fixed paraffin-embedded tissues were studied for DGCR8 immunoexpression. We present for the first time the p.(E518K) mutation in a case of poorly differentiated thyroid carcinoma and present the deregulation of DGCR8 expression at mRNA level in follicular-patterned tumours. The obtained data solidify DGCR8 as another important player of miRNA-related gene mutations in thyroid tumorigenesis, particularly in follicular-patterned thyroid tumours.