Search

In vitro transcription (IVT) is a DNA-templated process for synthesizing long RNA transcripts, including messenger RNA (mRNA). For many research and commercial applications, IVT of mRNA is typically performed using bacteriophage T7 RNA polymerase (T7 RNAP) owing to its ability to produce full-length RNA transcripts with high fidelity; however, T7 RNAP can also produce immunostimulatory byproducts such as double-stranded RNA that can affect protein expression. Such byproducts require complex purification processes, using methods such as reversed-phase high-performance liquid chromatography, to yield safe and effective mRNA-based medicines. To minimize the need for downstream purification processes, we rationally and computationally engineered a double mutant of T7 RNAP that produces substantially less immunostimulatory RNA during IVT compared with wild-type T7 RNAP. The resulting mutant allows for a simplified production process with similar mRNA potency, lower immunostimulatory content and quicker manufacturing time compared with wild-type T7 RNAP. Herein, we describe the computational design and development of this improved T7 RNAP variant. The purity of synthetic mRNA is improved with a double-mutant T7 RNA polymerase. [ABSTRACT FROM AUTHOR]

Preeclampsia is a placentally induced hypertensive disorder of pregnancy that is associated with substantial morbidity and mortality to mothers and fetuses. Clinical manifestations of preterm preeclampsia result from excess circulating soluble vascular endothelial growth factor receptor FLT1 (sFLT1 or sVEGFR1) of placental origin. Here we identify short interfering RNAs (siRNAs) that selectively silence the three sFLT1 mRNA isoforms primarily responsible for placental overexpression of sFLT1 without reducing levels of full-length FLT1 mRNA. Full chemical stabilization in the context of hydrophobic modifications enabled productive siRNA accumulation in the placenta (up to 7% of injected dose) and reduced circulating sFLT1 in pregnant mice (up to 50%). In a baboon preeclampsia model, a single dose of siRNAs suppressed sFLT1 overexpression and clinical signs of preeclampsia. Our results demonstrate RNAi-based extrahepatic modulation of gene expression with nonformulated siRNAs in nonhuman primates and establish a path toward a new treatment paradigm for patients with preterm preeclampsia. [ABSTRACT FROM AUTHOR]

Innate immune responses combat infectious microorganisms by inducing inflammatory responses, antimicrobial pathways and adaptive immunity. Multiple genes within each of these functional categories are coordinately and temporally regulated in response to distinct external stimuli. The substantial potential of these responses to drive pathological inflammation and tissue damage highlights the need for rigorous control of these responses. Although transcriptional control of inflammatory gene expression has been studied extensively, the importance of post-transcriptional regulation of these processes is less well defined. In this Review, we discuss the regulatory mechanisms that occur at the level of mRNA splicing, mRNA polyadenylation, mRNA stability and protein translation, and that have instrumental roles in controlling both the magnitude and duration of the inflammatory response. [ABSTRACT FROM AUTHOR]

Human Staufen1 (Stau1) is a double-stranded RNA (dsRNA)-binding protein implicated in multiple post-transcriptional gene-regulatory processes. Here we combined RNA immunoprecipitation in tandem (RIPiT) with RNase footprinting, formaldehyde cross-linking, sonication-mediated RNA fragmentation and deep sequencing to map Staufen1-binding sites transcriptome wide. We find that Stau1 binds complex secondary structures containing multiple short helices, many of which are formed by inverted Alu elements in annotated 3′ untranslated regions (UTRs) or in 'strongly distal' 3′ UTRs. Stau1 also interacts with actively translating ribosomes and with mRNA coding sequences (CDSs) and 3′ UTRs in proportion to their GC content and propensity to form internal secondary structure. On mRNAs with high CDS GC content, higher Stau1 levels lead to greater ribosome densities, thus suggesting a general role for Stau1 in modulating translation elongation through structured CDS regions. Our results also indicate that Stau1 regulates translation of transcription-regulatory proteins. [ABSTRACT FROM AUTHOR]

Amyotrophic lateral sclerosis (ALS) is a late-onset neurodegenerative disorder resulting from motor neuron death. Approximately 10% of cases are familial (FALS), typically with a dominant inheritance mode. Despite numerous advances in recent years, nearly 50% of FALS cases have unknown genetic aetiology. Here we show that mutations within the profilin 1 (PFN1) gene can cause FALS. PFN1 is crucial for the conversion of monomeric (G)-actin to filamentous (F)-actin. Exome sequencing of two large ALS families showed different mutations within the PFN1 gene. Further sequence analysis identified 4 mutations in 7 out of 274 FALS cases. Cells expressing PFN1 mutants contain ubiquitinated, insoluble aggregates that in many cases contain the ALS-associated protein TDP-43. PFN1 mutants also display decreased bound actin levels and can inhibit axon outgrowth. Furthermore, primary motor neurons expressing mutant PFN1 display smaller growth cones with a reduced F/G-actin ratio. These observations further document that cytoskeletal pathway alterations contribute to ALS pathogenesis. [ABSTRACT FROM AUTHOR]

Innate immune responses combat infectious microorganisms by inducing inflammatory responses, antimicrobial pathways and adaptive immunity. Multiple genes within each of these functional categories are coordinately and temporally regulated in response to distinct external stimuli. The substantial potential of these responses to drive pathological inflammation and tissue damage highlights the need for rigorous control of these responses. Although transcriptional control of inflammatory gene expression has been studied extensively, the importance of post-transcriptional regulation of these processes is less well defined. In this Review, we discuss the regulatory mechanisms that occur at the level of mRNA splicing, mRNA polyadenylation, mRNA stability and protein translation, and that have instrumental roles in controlling both the magnitude and duration of the inflammatory response. [ABSTRACT FROM AUTHOR]

The exon junction complex (EJC), a set of proteins deposited on mRNAs as a consequence of pre-mRNA splicing, is a key effector of downstream mRNA metabolism. We have identified eIF4AIII, a member of the eukaryotic translation initiation factor 4A family of RNA helicases (also known as DExH/D box proteins), as a novel EJC core component. Crosslinking and antibody inhibition studies suggest that eIF4AIII constitutes at least part of the platform anchoring other EJC components to spliced mRNAs. A nucleocytoplasmic shuttling protein, eIF4AIII associates in vitro and in vivo with two other EJC core factors, Y14 and Magoh. In mammalian cells, eIF4AIII is essential for nonsense-mediated mRNA decay (NMD). Finally, a model is proposed by which eIF4AIII represents a new functional class of DExH/D box proteins that act as RNA clamps or 'place holders' for the sequence-independent attachment of additional factors to RNAs. [ABSTRACT FROM AUTHOR]

The spliceosome is a multimegadalton RNA-protein machine that removes noncoding sequences from nascent pre-mRNAs. Recruitment of the spliceosome to splice sites and subsequent splicing require a series of dynamic interactions among the spliceosome's component U snRNPs and many additional protein factors. These dynamics present several challenges for structural analyses, including purification of stable complexes to compositional homogeneity and assessment of conformational heterogeneity. We have isolated spliceosomes arrested before the second chemical step of splicing (C complex) in which U2, U5 and U6 snRNAs are stably associated. Using electron microscopy, we obtained images of C complex spliceosomes under cryogenic conditions and determined a three-dimensional structure of a core complex to a resolution of 30 Å. The structure reveals a particle of dimensions 27 × 22 × 24 nm with a relatively open arrangement of three primary domains. [ABSTRACT FROM AUTHOR]

Provides evidence that lariat formation and exon ligation as transesterification reactions occur as single `in-line' SN2 nucleophilic displacement reactions. Excision of introns from nuclear precursors to messenger RNAs; Use of splicing substrates containing phosphorothioate; Site for spliceosomal processing.

The molecular basis for the consensus sequence at the 3' ends of introns in higher eukaryotes has now been elucidated. However, this discovery does not explain all aspects of 3' splice site selection. [ABSTRACT FROM AUTHOR]

Reports on the description of a gene in which all the functional sequences are seemingly contained within the introns. Tendency to ignore intronic sequences; Active functions of introns; Presence of functional RNAs within introns of genes whose exons specify proteins involved in translation.

Focuses on the in vitro RNA selection and evolution. Biochemical pathways for the synthesis of activated nucleosides; Recapitulation of the primordial ribozyme activities; Description of a self-aminoacylating RNA.