Tissue-specific consequences of tag fusions on protein expression in transgenic mice.

Genetic fusion of protein tags is widely used to study protein functions in vivo. It is well known that tag fusion can cause unwanted changes in protein stability, but whether this is an inherent property of the tagged protein, or can be influenced by the cell and tissue environment, is unclear. Using a series of genome edited mouse models, we show that tag-dependent changes in protein expression can vary across different primary cell and tissue contexts.

Modeling flexible RNA 3D structures and RNA-protein complexes.

RNA and RNA-protein (RNP) complexes are central to many cellular processes, but the determination of their structures remains challenging due to RNA flexibility and interaction diversity. This review highlights recent computational advances, particularly from the past two years, in predicting and analyzing RNA and RNP structures. We discuss template-based modeling, docking, molecular simulations, and deep learning approaches, with an emphasis on emerging hybrid methods that integrate these strategies.

ARTEM: a method for RNA and DNA tertiary motif identification with backbone permutations.

Non-coding RNA functions are largely defined by their 3D structures, which consist of recurrent building blocks, tertiary motifs. The computational motif search problem remains largely unsolved, as standard approaches are restrained by sequence, interactions, or backbone topology. We present ARTEM 2.

Structure of fungal tRNA ligase Trl1 with RNA reveals conserved substrate-binding principles.

RNA ligases play a vital role in RNA processing and maturation, including tRNA splicing, RNA repair and the unfolded protein response (UPR). In fungi and plants, the tripartite tRNA ligase Trl1 catalyzes the joining of TSEN-cleaved pre-tRNA exon halves. Trl1 also functions as ligase in the non-conventional HAC1 mRNA splicing during the UPR.

The expanding repertoire of ESCRT functions in cell biology and disease.

The endosomal sorting complex required for transport (ESCRT) is a multicomplex machinery comprising proteins that are conserved from bacteria to humans and has diverse roles in regulating the dynamics of cellular membranes. ESCRT functions have far-reaching consequences for cell biological processes such as intracellular traffic, membrane repair, cell signalling, metabolic regulation, cell division and genome maintenance. Here we review recent insights that emphasize the pathophysiological consequences of ESCRT dysfunctions, including infections, immune disorders, cancers and neurological diseases.

Autophagy at the postsynapse begins with Rab11 and does not end with dendritic spine pruning.

Neurons are highly differentiated and compartmentalized cells that conduct cellular processes in a spatiotemporally regulated manner. Autophagy in neurons occurs locally under stimulation and contributes to synaptic plasticity. Little is known about the initial steps leading to autophagy upon neuronal stimulation and the role of autophagic compartments at the postsynaptic part of the synapse.

Introducing the "other" type of DNA methylation.

De novo adenine DNA methyltransferases have been identified in the ciliate .

Activity and structure of human (d)CTP deaminase CDADC1.

Vertebrates have evolved an understudied protein termed CDADC1 (NYD-SP15) that contains an inactive N-terminal and active C-terminal DCTD-like domain. Here, we show that human CDADC1 is a (d)CTP-specific deaminase, with a roughly 2-fold in vitro preference for dCTP over CTP. We determined high-resolution cryo-EM structures of CDADC1 in the absence of substrate and in complex with dCTP and 5-methyl-dCTP.

Determining the effects of pseudouridine incorporation on human tRNAs.

Transfer RNAs (tRNAs) are ubiquitous non-coding RNA molecules required to translate mRNA-encoded sequence information into nascent polypeptide chains. Their relatively small size and heterogenous patterns of their RNA modifications have impeded the systematic structural characterization of individual tRNAs. Here, we use single-particle cryo-EM to determine the structures of four human tRNAs before and after incorporation of pseudouridines (Ψ).

Protein-RNA Docking Benchmark v3.0 Integrated With Binding Affinity.

We introduce an updated non-redundant protein-RNA docking benchmark version 3.0 (PRDBv3.0) containing 197 test cases curated from 288 unique protein-RNA complexes available in the Protein Data Bank until July 2024.

Statistical framework for calling allelic imbalance in high-throughput sequencing data.

High-throughput sequencing facilitates large-scale studies of gene regulation and allows tracing the associations of individual genomic variants with changes in gene regulation and expression. Compared to classic association studies, the assessment of an allelic imbalance at heterozygous variants captures functional variant effects with smaller sample sizes, higher sensitivity, and better resolution. Yet, identification of allele-specific variants from allelic read counts remains challenging due to data-dependent biases and overdispersion arising from technical and biological variability.

DIS3L, cytoplasmic exosome catalytic subunit, is essential for development but not cell viability in mice.

Among numerous enzymes involved in RNA decay, processive exoribonucleases are the most prominent group responsible for the degradation of entire RNA molecules. The role of mammalian cytoplasmic 3'-5' exonuclease DIS3L at the organismal level remained unknown. Herein, we established knock-in and knockout (KO) mouse models to study DIS3L functions in mice.

Role of Non-Canonical Stacking Interactions of Heterocyclic RNA Bases in Ribosome Function.

Identification and analysis of repetitive elements (motifs) in DNA, RNA, and protein macromolecules is an important step in studying structure and functions of these biopolymers. Functional role of NA-BSE (non-adjacent base-stacking element, a widespread tertiary structure motif in various RNAs) in RNA-RNA interactions at various stages of the ribosome function during translation has been investigated in this work. Motifs of this type have been described to date that are reversibly formed during mRNA decoding, moving of the ribosome subunits relative to each other, and moving mRNA and tRNA along the ribosome during translocation.

Effects of genetic ablation and pharmacological inhibition of HuR on gene expression, iron metabolism, and hormone levels.

Background: HuR/ELAV1, a ubiquitous RNA-binding protein, belongs to the RNA-binding protein family and is crucial for stabilizing and regulating the translation of various mRNA targets, influencing gene expression. Elevated HuR levels are associated with multiple disorders, including cancer and neurodegenerative diseases. Despite the identification of small molecule inhibitors targeting HuR, their detailed characterization remains limited.

Computational analysis of congenital heart disease associated SNPs: unveiling their impact on the gene regulatory system.

Congenital heart disease (CHD) is a prevalent condition characterized by defective heart development, causing premature death and stillbirths among infants. Genome-wide association studies (GWASs) have provided insights into the role of genetic variants in CHD pathogenesis through the identification of a comprehensive set of single-nucleotide polymorphisms (SNPs). Notably, 90-95% of these variants reside in the noncoding genome, complicating the understanding of their underlying mechanisms.

DesiRNA: structure-based design of RNA sequences with a replica exchange Monte Carlo approach.

Designing RNA sequences that form a specific structure remains a challenge. Current computational methods often struggle with the complexity of RNA structures, especially when considering pseudoknots or restrictions related to RNA function. We developed DesiRNA, a computational tool for the design of RNA sequences based on the Replica Exchange Monte Carlo approach.

Protocol for the preparation of zebrafish whole heart cell suspension for single-cell analyses.

Obtaining viable cell suspension that accurately represents the diversity of complex tissues is challenging due to the distinct characteristics of each cell type. Here, we present a protocol for preparing a single-cell suspension of the zebrafish embryonic whole heart, detailing steps for heart extraction, cell dissociation, quantification, and quality assessment. This suspension is compatible with downstream analysis on various single-cell platforms.

Protocol for the visualization of pRps6-positive cells in larval zebrafish brains using whole-mount immunofluorescence and light-sheet microscopy.

Due to their small size and transparency, larval zebrafish are a useful model for whole-brain imaging. Here, we present a protocol for the visualization of phosphorylated Rps6, a marker of mechanistic target of rapamycin complex 1 (mTORC1) activity, in the zebrafish brains at 5 days post fertilization (dpf), using whole-mount immunofluorescence and light-sheet microscopy. We describe steps for sample preparation, storage, staining, and imaging.

Protocol for microinjection of rapamycin into the zebrafish habenula.

Mechanistic target of rapamycin complex 1 (mTorC1) activity plays a crucial role in brain development. Here, we present an approach for rapamycin microinjection into the habenula of larval zebrafish to achieve localized inhibition of the mTorC1 pathway and explore the role of mTorC1 in habenula function. We describe steps for performing microinjections and maintaining zebrafish larvae before and after the procedure.

5' terminal nucleotide determines the immunogenicity of IVT RNAs.

In vitro transcription (IVT) is a technology of vital importance that facilitated the production of mRNA therapeutics and drove numerous breakthroughs in RNA biology. T7 polymerase-produced RNAs can begin with either 5'-triphosphate guanosine (5'-pppG) or 5'-triphosphate adenosine (5'-pppA), generating potential agonists for the RIG-I/type I interferon response. While it is established that IVT can yield highly immunogenic double-stranded RNA (dsRNA) via promoterless transcription, the specific contribution of initiating nucleosides to this process has not been previously reported.

Data storage based on the absence of nucleotides using a bacteriophage abortive infection system reverse transcriptase.

DNA molecules are a promising data storage medium for the future; however, effective synthesis of DNA using an enzyme that catalyzes the polymerization of natural nucleoside triphosphates in a user-defined manner, without the need for multiple injections of polymerase, remains a challenge. In the present study, we demonstrated that the bacteriophage abortive infection system reverse transcriptase AbiK from facilitates such an approach. We employed surface plasmon resonance to monitor the polymerization of the DNA strand with a user-defined sequence of multiple segments through a sequential buffer exchange process.

Impact of water models on the structure and dynamics of enzyme tunnels.

Protein hydration plays a vital role in many biological functions, and molecular dynamics simulations are frequently used to study it. However, the accuracy of these simulations is often sensitive to the water model used, a phenomenon particularly evident in intrinsically disordered proteins. Here, we investigated the extent to which the choice of water model alters the behavior of complex networks of tunnels within proteins.