Evidence that immune RNA is messenger RNA.

Exposure of rabbit spleen cell cultures to i-RNA isolated from T2 phage-exposed rabbit peritoneal exudate cells induces the synthesis of antigen and allotype specific 19S proteins even in the presence of actinomycin D. The same i-RNA directs the synthesis of proteins with comparable properties in cell-free extracts prepared from mouse L cells, indicating that i-RNA functions as mRNA and contains the information required to code for the synthesis of IgM antibodies.     

Monocistronic messenger RNA in yeast

We have determined the rate of polypeptide chain synthesis on different size polysomes in yeast. The completion time for the average polypeptide chain in vivo at 23 °C is two minutes by this technique and is in good agreement with values we have determined by other independent methods.These kinetic experiments indicate that the average size of a nascent polypeptide chain on a polysome is directly related to the size of the polysome. This demonstrates that in the simple eucaryotic organism, Saccharomyces cerevisiae, mRNA is monocistronic in the sense that each mRNA molecule codes for one protein molecule which is released intact from the ribosome upon completion. The pattern of amino acid incorporation into Escherichia coli polysomes is distinctly different. These findings have a number of interesting implications for the genetics of the lower eucaryotes and indicate that the cellular mechanisms of control and co-ordination in yeast may differ from those found in procaryotes and may be similar to cellular mechanisms of control for mammalian cells.     

Autoimmune epitopes in messenger RNA

Patients with systemic autoimmune disorders produce autoantibodies against sequence-specific conformational RNA epitopes on U1 snRNA, 28S rRNA, and transfer RNAs. The molecular basis for immunological reactivity with these highly abundant and stable RNAs is not understood. Here, we report the existence of discrete RNA epitopes in messenger RNAs that are generally less abundant and less stable than snRNAs and tRNAs. An iterative selection and amplification procedure using pooled autoimmune patient sera identified immunoreactive mRNA species. Following deconvolution of the pools to identify the reactive sera, several mRNAs recognized by these autoantibodies were cloned and sequenced. Detailed analysis using one particular serum indicated reactivity against the messages encoding alternative splicing factor (ASF/SF2) and calmodulin. Deletion and site-directed mutagenesis determined that an epitope recognized by this serum is located in a 17-base stem-loop structure common to both messages. This serum was then used to immunoprecipitate native mRNAs encoding ASF/SF2 and calmodulin from total HeLa cell RNA. Our results demonstrate that despite its low abundance and instability, messenger RNA is capable of reacting with autoantibodies generated during an autoimmune response. These data are consistent with direct presentation as a model to explain the generation of RNA conformation-specific autoantibodies.     

A precursor of globin messenger RNA

The size of pulse-labeled globin messenger RNA nucleotide sequences was investigated, to determine whether newly transcribed globin mRNA molecules are larger than steady-state globin mRNA. Molecular hybridization techniques were used to compare directly the sedimentation of steady-state (unlabeled) and pulse-labeled (radioactive) globin mRNA sequences in the same analytical sucrose gradient. In gradients containing 98% formamide, radioactive globin mRNA sequences from mouse fetal liver cells labeled for 15 to 20 minutes with [³H]uridine sediment in a broad band with a peak at approximately 14 S, while steady-state globin mRNA sediments at 10 S. The large radioactive RNA can be recovered from one gradient and recentrifuged in a second gradient, in which it again sediments in a broad band with a peak at 14 S. The large radioactive RNA is cleaved to 10 S during a 75-minute “chase” with either actinomycin D or unlabeled uridine plus cytidine. The estimated half-life of the precursor is 45 minutes or less under these conditions. A covalent RNA precursor larger than 18 S with a similar turnover rate is not observed.     

Translational capacity of deadenylated messenger RNA

In order to determine whether the functional capacity of polyadenylated messenger RNA is altered when the poly(A) segment is shortened as a consequence of mRNA aging, we compared the abilities of differentially labeled new and old mRNAs to engage ribosomes. Experiments were performed using both exponentially growing mouse L cells and L cells in which the polyribosomes were temporarily disassembled as a result of a temperature shock. Our results indicate that new and old messages engage ribosomes with roughly equivalent efficiencies, suggesting, therefore, that length of poly(A) is not a determining factor for mRNA translation. We then examined the question of whether an mRNA that was originally polyadenylated could function if the Poly(A) segment were removed. This was accomplished by submitting polyadenylated mRNA to a limited digestion with a 3′-OH specific exoribonuclease, reisolating the residual polyadenylated and deadenylated mRNA, and measuring their template activities using a cell-free protein synthesizing system prepared from wheat embryos. Such experiments also failed to reveal any effect of the poly(A) on the translational capacity of the mRNA.     

Translation and messenger RNA secondary structure

The possibility of translation being influenced by the messenger RNA secondary structure is investigated with the aid of a stochastic model. Simulations indicate that, at least for certain mRNA's, the mean ribosomal passage time decreases as the mean number of ribosomes on the messenger is increased. Furthermore, large variations in the passage times are found, in accordance with recent experimental results.     

Minimal methylation of yeast messenger RNA

Most, if not all, yeast mRNAs are capped at their 5-terminus by m⁷G. Apart from m⁷G no other methylated nucleotides could be detected in poly (A)⁺ mRNA isolated from yeast polysomes.Abbreviations used poly (A)⁺ mRNA messenger RNA containing poly (A) - poly (A)^- RNA RNA lacking poly (A) - m⁷G N₇-methyl guanosine - N_m any 2-0 methylated nucleoside - mN any basemethylated nucleoside