An RNA exosome subunit mediates cell-to-cell trafficking of a homeobox mRNA via plasmodesmata

mRNA migration through plasmodesmata In plants, certain transcription factors are produced in one cell but transported, sometimes as messenger RNA (mRNA), through plasmodesmata, channels between neighboring plant cells, where they act. This system helps to manage stem cell development. Kitagawa et al . now identify part of the machinery that manages this cell-to-cell transport. Transport of the mRNA encoding the KNOTTED1 homeobox transcription factor depends on Ribosomal RNA-Processing Protein 44 (AtRRP44A), which is a subunit of the RNA exosome. —PJH

The Thiazole-5-Carboxamide GPS491 Inhibits HIV-1, Adenovirus, and Coronavirus Replication by Altering RNA Processing/Accumulation

Medicinal chemistry optimization of a previously described stilbene inhibitor of HIV-1, 5350150 (2-(2-(5-nitro-2-thienyl)vinyl)quinoline), led to the identification of the thiazole-5-carboxamide derivative (GPS491), which retained potent anti-HIV-1 activity with reduced toxicity. In this report, we demonstrate that the block of HIV-1 replication by GPS491 is accompanied by a drastic inhibition of viral gene expression (IC50 ~ 0.25 µM), and alterations in the production of unspliced, singly spliced, and multiply spliced HIV-1 RNAs. GPS491 also inhibited the replication of adenovirus and multiple coronaviruses. Low µM doses of GPS491 reduced adenovirus infectious yield ~1000 fold, altered virus early gene expression/viral E1A RNA processing, blocked viral DNA amplification, and inhibited late (hexon) gene expression. Loss of replication of multiple coronaviruses (229E, OC43, SARS-CoV2) upon GPS491 addition was associated with the inhibition of viral structural protein expression and the formation of virus particles. Consistent with the observed changes in viral RNA processing, GPS491 treatment induced selective alterations in the accumulation/phosphorylation/function of splicing regulatory SR proteins. Our study establishes that a compound that impacts the activity of cellular factors involved in RNA processing can prevent the replication of several viruses with minimal effect on cell viability.

Cofactor-Independent RNA Editing by a Synthetic S-Type PPR Protein

ABSTRACT Pentatricopeptide repeat (PPR) proteins are RNA-binding proteins that are attractive tools for RNA processing in synthetic biology applications given their modular structure and ease of design. Several distinct types of motifs have been described from natural PPR proteins, but almost all work so far with synthetic PPR proteins has focused on the most widespread P-type motifs. We have investigated synthetic PPR proteins based on tandem repeats of the more compact S-type PPR motif found in plant organellar RNA editing factors, and particularly prevalent in the lycophyte Selaginella. With the aid of a novel plate-based screening method we show that synthetic S-type PPR proteins are easy to design, bind with high affinity and specificity, and are functional in a wide range of pH, salt and temperature conditions. We find that they outperform a synthetic P-type PPR scaffold in many situations. We designed an S-type editing factor to edit an RNA target in E. coli and demonstrate that it edits effectively without requiring any additional cofactors to be added to the system. These qualities make S-type PPR scaffolds ideal for developing new RNA processing tools.

Mapping information-rich genotype-phenotype landscapes with genome-scale Perturb-seq

A central goal of genetics is to define the relationships between genotypes and phenotypes. High-content phenotypic screens such as Perturb-seq (pooled CRISPR-based screens with single-cell RNA-sequencing readouts) enable massively parallel functional genomic mapping but, to date, have been used at limited scales. Here, we perform genome-scale Perturb-seq targeting all expressed genes with CRISPR interference (CRISPRi) across >2.5 million human cells and present a framework to power biological discovery with the resulting genotype-phenotype map. We use transcriptional phenotypes to predict the function of poorly-characterized genes, uncovering new regulators of ribosome biogenesis (including CCDC86, ZNF236, and SPATA5L1), transcription (C7orf26), and mitochondrial respiration (TMEM242). In addition to assigning gene function, single-cell transcriptional phenotypes allow for in-depth dissection of complex cellular phenomena - from RNA processing to differentiation. We leverage this ability to systematically identify the genetic drivers and consequences of aneuploidy and to discover an unanticipated layer of stress-specific regulation of the mitochondrial genome. Our information-rich genotype-phenotype map reveals a multidimensional portrait of gene function and cellular behavior.

A Novel Chloroplast Protein RNA Processing 8 Is Required for the Expression of Chloroplast Genes and Chloroplast Development in Arabidopsis thaliana

Chloroplast development involves the coordinated expression of both plastids- and nuclear-encoded genes in higher plants. However, the underlying mechanism still remains largely unknown. In this study, we isolated and characterized an Arabidopsis mutant with an albino lethality phenotype named RNA processing 8 (rp8). Genetic complementation analysis demonstrated that the gene AT4G37920 (RP8) was responsible for the mutated phenotype. The RP8 gene was strongly expressed in photosynthetic tissues at both transcription and translation protein levels. The RP8 protein is localized in the chloroplast and associated with the thylakoid. Disruption of the RP8 gene led to a defect in the accumulation of the rpoA mature transcript, which reduced the level of the RpoA protein, and affected the transcription of PEP-dependent genes. The abundance of the chloroplast rRNA, including 23S, 16S, 4.5S, and 5S rRNA, were reduced in the rp8 mutant, respectively, and the amounts of chloroplast ribosome proteins, such as, PRPS1(uS1c), PRPS5(uS5c), PRPL2 (uL2c), and PRPL4 (uL4c), were substantially decreased in the rp8 mutant, which indicated that knockout of RP8 seriously affected chloroplast translational machinery. Accordingly, the accumulation of photosynthetic proteins was seriously reduced. Taken together, these results indicate that the RP8 protein plays an important regulatory role in the rpoA transcript processing, which is required for the expression of chloroplast genes and chloroplast development in Arabidopsis.

RNA Binding Protein SRSF3 confers an essential role in megakaryocyte maturation and platelet production

RNA processing is increasingly recognised as a critical control point in the regulation of different haematopoietic lineages including megakaryocytes responsible for the production of platelets. Platelets are anucleate cytoplasts that contain a rich repertoire of RNAs encoding proteins with essential platelet functions derived from the parent megakaryocyte. It is largely unknown how RNA binding proteins contribute to the development and functions of megakaryocytes and platelets. We show that Serine-arginine rich splicing factor 3 (SRSF3) is essential for megakaryocyte maturation and generation of functional platelets. Megakaryocyte-specific deletion of Srsf3 in mice led to macrothrombocytopenia characterised by megakaryocyte maturation arrest, dramatically reduced platelet counts and abnormally large functionally compromised platelets. SRSF3 deficient megakaryocytes failed to reprogram their transcriptome during maturation and to load platelets with RNAs required for normal platelet function. SRSF3 depletion led to nuclear accumulation of megakaryocyte mRNAs demonstrating that SRSF3 deploys similar RNA regulatory mechanisms in megakaryocytes as in other cell types. Our study further suggests that SRSF3 plays a role in sorting cytoplasmic megakaryocyte RNAs into platelets and demonstrates how SRSF3-mediated RNA processing forms a central part of megakaryocyte gene regulation. Understanding SRSF3 functions in megakaryocytes and platelets provides key insights into normal thrombopoiesis and platelet pathologies as SRSF3 RNA targets in megakaryocytes are associated with platelet diseases.

Intricate crosstalk between MYB and noncoding RNAs in cancer

MYB is often overexpressed in malignant tumors and plays a carcinogenic role in the initiation and development of cancer. Deletion of the MYB regulatory C-terminal domain may be a driving mutation leading to tumorigenesis, therefore, different tumor mechanisms produce similar MYB proteins. As MYB is a transcription factor, priority has been given to identifying the genes that it regulates. All previous attention has been focused on protein-coding genes. However, an increasing number of studies have suggested that MYB can affect the complexity of cancer progression by regulating tumor-associated noncoding RNAs (ncRNAs), such as microRNAs, long-non-coding RNAs and circular RNAs. ncRNAs can regulate the expression of numerous downstream genes at the transcription, RNA processing and translation levels, thereby having various biological functions. Additionally, ncRNAs play important roles in regulating MYB expression. This review focuses on the intricate crosstalk between oncogenic MYB and ncRNAs, which play a pivotal role in tumorigenesis, including proliferation, apoptosis, angiogenesis, metastasis, senescence and drug resistance. In addition, we discuss therapeutic strategies for crosstalk between MYB and ncRNAs to prevent the occurrence and development of cancer.

A shape-shifting nuclease unravels structured RNA

RNA turnover pathways ensure appropriate gene expression levels by eliminating unwanted transcripts that may otherwise interfere with cellular programs. The enzyme Dis3-like protein 2 (Dis3L2) is a 3′-5′ exoribonuclease that, through its RNA turnover activity, plays a critical role in human development1. Dis3L2 can independently degrade structured substrates and its targets include many coding and non-coding 3′-uridylated RNAs1-5. While the basis for Dis3L2 substrate recognition has been well-characterized6, the mechanism of structured RNA degradation by this family of enzymes is unknown. We characterized the discrete steps of the degradation cycle by determining electron cryo-microscopy structures representing snapshots along the RNA turnover pathway and measuring kinetic parameters for single-stranded (ss) and double-stranded (ds) RNA processing. We discovered a dramatic conformational change that is triggered by the dsRNA, involving repositioning of two cold shock domains by 70 Å. This movement exposes a trihelix-linker region, which acts as a wedge to separate the two RNA strands. Furthermore, we show that the trihelix linker is critical for dsRNA, but not ssRNA, degradation. These findings reveal the conformational plasticity of this enzyme, and detail a novel mechanism of structured RNA degradation.