The structure of pre-messenger RNA and messenger RNA from erythroid cells

Pre-mRNA fractions (greater than 45 S) were characterized by electron microscopy. High salt concentrations (0.2 M ammonium acetate, pH 8) yield linear molecules of different length (0.5--17 micrometer). In 10% of the molecules a compact-nonlinear contour (cn-contour) is detectable at one end. A significant enhancement of the number of cn-contour carrying molecules is observed after binding pre-mRNA to poly(U)-sepharose. The terminal cn-contour could be the depiction of a secondary and/or tertiary structure including the poly(A)-tail. 9 S globin mRNA appear in 80% with virtually the same cn-contour as detected in pre-mRNA molecules. After denaturing the mRNA in 80% formamide--4M urea in connection with heating to 90 degrees C from 10 min, a percentage of 77% of stretched, linear molecules results. This structural transformation is reversible when the denatured RNA is precipitated and redissolved in 0.2 M ammonium acetate. 73% of the stretched molecules are characterized by a mean length of 0.44 micrometer. This value is twice as high as commonly assumed for a globin mRNA chain.     

Frequency distribution of pre-messenger RNA sequences in polyadenylated and non-polyadenylated nuclear RNA from Friend cells.

Hybridisation of cDNA probes for abundant and rare polysomal polyadenylated RNAs with polyadenylated and non-polyadenylated nuclear RNA from Friend cells indicated that the abundant polysomal polyadenylated RNA sequences were present at a higher concentration in the nucleus than rare polysomal sequences, but at a reduced range of concentrations. The ratio of the concentrations of abundant and rare sequences was about 3 in non-polyadenylated nuclear RNA, 9 in polyadenylated nuclear RNA and 13 in polysomal polyadenylated RNA. This suggests that polyadenylation may play a role in the quantitative selection of sequences for transport to the cytoplasm. Polyadenylation cannot be the only signal for transport, since a highly complex population of nucleus-confined polyadenylated molecules exists, each of which is present on average at less than one copy per cell.     

Trans splicing of nematode pre-messenger RNA in vitro

In nematodes, a fraction of mRNAs contains a common 22 nucleotide 5' terminal spliced leader (SL) sequence derived by trans splicing. Here, we show that a cell-free extract prepared from developing embryos of the parasitic nematode Ascaris lumbricoides catalyzes accurate and efficient SL addition to a synthetic pre-mRNA at an authentic trans splice acceptor site. SL addition occurs via a trans splicing reaction that proceeds through Y-branched intermediates. The branchpoint is located at either of two adenosine residues located 18 and 19 nucleotides upstream of the splice acceptor site.     

Identification and nuclear localization of yeast pre-messenger RNA processing components: RNA2 and RNA3 proteins

Temperature-sensitive mutations in the RNA2 through RNA11 genes of yeast prevent the processing of nuclear pre-mRNAs. We have raised antisera that detect the RNA2 and RNA3 proteins in immunoblots of extracts of yeast containing high copy number RNA2 and RNA3 plasmids. Subcellular fractionation of yeast cells that overproduce the RNA2 and RNA3 proteins has revealed that these proteins are enriched in nuclear fractions. Indirect immunofluorescence results have indicated that these proteins are localized in yeast nuclei. These localization results are consistent with the fact that these genes have a role in processing yeast pre-mRNA.     

Engineered exosome-mediated messenger RNA and single-chain variable fragment delivery for human chimeric antigen receptor T-cell engineering

Background aimsMost current chimeric antigen receptor (CAR) T cells are generated by viral transduction, which induces persistent expression of CARs and may cause serious undesirable effects. Messenger RNA (mRNA)-based approaches in manufacturing CAR T cells are being developed to overcome these challenges. However, the most common method of delivering mRNA to T cells is electroporation, which can be toxic to cells.MethodsThe authors designed and engineered an exosome delivery platform using the bacteriophage MS2 system in combination with the highly expressed protein lysosome-associated membrane protein 2 isoform B on exosomes.ResultsThe authors’ delivery platform achieved specific loading and delivery of mRNA into target cells and achieved expression of specific proteins, and anti-CD3/CD28 single-chain variable fragments (scFvs) expressed outside the exosomal membrane effectively activated primary T cells in a similar way to commercial magnetic beads.ConclusionsThe delivery of CAR mRNA and anti-CD3/CD28 scFvs via designed exosomes can be used for ex vivo production of CAR T cells with cancer cell killing capacity. The authors’ results indicate the potential applications of the engineered exosome delivery platform for direct conversion of primary T cells to CAR T cells while providing a novel strategy for producing CAR T cells in vivo.     

Degradation of pre-messenger RNA in bovine thyroid slices; effect of thyrotropin

Summary Bovine thyroid RNA labeled by incubation of slices in the presence of ³²P-orthophosphate were fractionated by a two-step procedure. Total RNA were extracted by gel filtration on AcA 22 in the presence of pronase and separated by Sepharose 2B chromatography. A small fraction of heavily-labeled RNA (giant RNA) was obtained in the void volume (peak I); the major fraction of RNA (smaller than 45 S) was retarded on the column (peak II) and had a low specific radioactivity. Labeled and total RNA of peak I and labeled RNA species of peak II had a DNA-like nucleotide composition and were polyadenylated. In contrast, the nucleotide composition of total RNA of peak II was similar to that of ribosomal RNA and had a very low poly (adenylic acid) content. Pulse-chase experiments showed a precursor-product relationship between the two RNA fractions. These data indicate that labeled RNA of peak I and peak II likely correspond to newly-synthetized pre-mRNA and mRNA, respectively. Thyrotropin induced a decrease in the amount of ³²P-labeled pre-mRNA and a proportional increase of ³²P-labeled mRNA suggesting a stimulatory effect of the hormone on the degradation of pre-mRNA.Abbreviations SDS sodium dodecyl sulfate - TIPNS triisopropylnaphthalene disulfonic acid, sodium salt - TSH thyrotropin-stimulating hormone     

An NMD pathway in yeast involving accelerated deadenylation and exosome-mediated 3'-->5' degradation

Eukaryotic mRNAs containing premature termination codons are subjected to accelerated turnover, known as nonsense-mediated decay (NMD). Recognition of translation termination events as premature requires a surveillance complex, which includes the RNA helicase Upf1p. In Saccharomyces cerevisiae, NMD provokes rapid decapping followed by 5'-->3' exonucleolytic decay. Here we report an alternative, decapping-independent NMD pathway involving deadenylation and subsequent 3'-->5' exonucleolytic decay. Accelerated turnover via this pathway required Upf1p and was blocked by the translation inhibitor cycloheximide. Degradation of the deadenylated mRNA required the Rrp4p and Ski7p components of the cytoplasmic exosome complex, as well as the putative RNA helicase Ski2p. We conclude that recognition of NMD substrates by the Upf surveillance complex can target mRNAs to rapid deadenylation and exosome-mediated degradation.     

Reprogramming alternative pre-messenger RNA splicing through the use of protein-binding antisense oligonucleotides

Alternative pre-messenger RNA splicing is a major contributor to proteomic diversity in higher eukaryotes and represents a key step in the control of protein function in a large variety of biological systems. As a means of artificially altering splice site choice, we have investigated the impact of positioning proteins in the vicinity of 5' splice sites. We find that a recombinant GST-MS2 protein interferes with 5' splice site use, most efficiently when it binds upstream of that site. To broaden the use of proteins as steric inhibitors of splicing, we have tested the activity of antisense oligonucleotides carrying binding sites for the heterogeneous nuclear ribonucleoprotein A1/A2 proteins. In a HeLa cell extract, tailed oligonucleotides complementary to exonic sequences elicit strong shifts in 5' splice site selection. In four different human cell lines, an interfering oligonucleotide carrying A1/A2 binding sites also shifted the alternative splicing of the Bcl-x pre-mRNA more efficiently than oligonucleotides acting through duplex formation only. The use of protein-binding oligonucleotides that interfere with U1 small nuclear ribonucleoprotein binding therefore represents a novel and powerful approach to control splice site selection in cells.