Multiple messenger RNA species give rise to the structural diversity in acetylcholinesterase

Acetylcholinesterase exists predominantly as a secreted enzyme which remains cell-associated at specific extracellular locations. Its extensive structural diversity appears responsible for the unique cellular disposition of the enzyme. To examine the molecular basis of the structural divergence of acetylcholinesterase species, we hybridized total RNA from Torpedo californica electric organ with restriction fragments from a cDNA encoding the catalytic subunits of asymmetric species of acetylcholinesterase. Multiple RNA species up to 14 kilobases in length can be detected on Northern blots using a full-length cDNA for hybridization. Each of these RNA species also hybridizes with smaller restriction fragments within the open reading frame and 3'-untranslated region of the cDNA. This indicates that the entire open reading frame plus the 3'-untranslated region is contained in the large RNA species. RNase protection experiments revealed at least three points of divergence for the message species. One occurs within the COOH-terminal portion of the open reading frame at a position just 5' to the TGA stop codon. This divergence accounts for the two classes of acetylcholinesterase found in abundance in Torpedo. The site of splicing has been further defined by isolating a genomic clone containing the exon serving as the potential splice donor. We find a divergence between the cDNA and genomic DNA at the position estimated by the protection experiments. A less abundant divergence in mRNA can also be detected in the 3'-untranslated region. Another divergence occurs as a deleted sequence within the 5'-noncoding region and may be important for controlling translation efficiency. Since it is hypothesized that a single gene encodes acetylcholinesterase, the divergences in the very 3' region of the open reading frame and the 5'-noncoding region correspond to presumed splice junction boundaries where alternative RNA splicing occurs.     

Differential stability of trp messenger RNA synthesized originating at the trp promoter and pL promoter of lambda trp phage.

The trp operon translocated into the early region of phage λ can be transcribed under the control of two promoters, the authentic trp promoter (p_Ttrp mRNA) and the p_L promoter of the N gene (p_Ltrp mRNA) (Imamoto &; Tani, 1972; Segawa &; Imamoto, 1974). The p_Ltrp mRNA has a 5′-terminal λ N message. The functional and chemical stability of trp segments in these mRNA species have been assayed. To determine trp mRNA from λtrp, appropriate φ80trp DNAs were used as a DNA complement in DNA-RNA hybridization assays.When formation of mRNA is inhibited, the capacity to serve as template for enzyme synthesis decays at a comparable rate for p_L and p_Ttrp mRNA, and p_Ltrp mRNA seems to be translated as efficiently as is the normal p_Ttrp mRNA. In contrast to this similar functional stability, p_Ltrp mRNA shows a more than tenfold greater chemical stability than p_Ttrp mRNA. (p_Ttrp mRNA is degraded at the same rate as trp mRNA in uninfected bacteria. Bulk host mRNA also decays at its normal rate in cells infected with λtrp.)On the basis of those and more extensive experiments including the sedimentation analysis of those stabilized trp mRNA molecules, it is inferred that (1) the rate-limiting step to initiate bulk mRNA degradation is determined by a sequence located at or near the 5′ end of the messenger RNA; and (2) functional inactivation of each messenger is regulated independently of bulk chemical degradation of the message.Stabilization of the trp mRNA produced from the p_L promoter increases with time after phage infection. Thus, the stabilization requires a modification of the decay trigger, possibly by a phage-specific protein such as a nuclease or the N and/or tof gene that might bind to the mRNA.     

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.     

Prothymosin alpha enhances human and murine MHC class II surface antigen expression and messenger RNA accumulation.

Prothymosin alpha (ProT alpha) is an acidic polypeptide with potentiating effects on HLA-DR-restricted in vitro cellular immune response systems such as T cell proliferative responses to soluble proteins and cellular auto- or alloantigens. Experiments were performed to investigate the effect of ProT alpha on MHC class II Ag expression in human monocytes, murine splenocytes, and tumor cell lines at both protein and molecular levels. RIA and immunofluorescence analysis revealed that ProT alpha enhances HLA-DR surface Ag expression whereas Northern blot analysis demonstrated that ProT alpha causes significant accumulation of MHC class II mRNA. The enhancing effect of ProT alpha was demonstrated convincingly using precultured human peripheral monocytes, which are known to express decreased amounts of surface HLA-DR Ag, and HLA-DR-positive human cell lines. Moreover, ProT alpha was shown to induce HLA-DR Ag expression in a priori HLA-DR-negative tumor cells. Furthermore, ProT alpha was shown to be active in vivo. Splenocytes from mice pretreated with ProT alpha expressed more surface Ilpha Ag and contained more I alpha-specific mRNA. These findings suggest that ProT alpha may be important in the regulation of the immune response by enhancing MHC class II Ag expression in APC.     

Viral genome RNA serves as messenger early in the infectious cycle of murine leukemia virus.

When NIH/3T3 mouse fibroblasts were infected with the Moloney strain of murine leukemia virus, part of the viral genome RNA molecules were detected in polyribosomes of the infected cells early in the infectious cycle. The binding appears to be specific, since we could demonstrate the release of viral RNA from polyribosomes with EDTA. Moreover, when infection occurred in the presence of cycloheximide, most viral RNA molecules were detected in the free cytoplasm. Size analysis on polyribosomal viral RNA molecules indicated that two size class molecules, 38S and 23S, are present in polyribosomes at 3 h after infection. Analysis of the polyriboadenylate [poly(rA)] content of viral RNA extracted from infected polyribosomes demonstrated that such molecules bind with greatest abundance at 3 h after infection, as has been detected with total viral RNA. No molecules lacking poly(rA) stretches could be detected in polyribosomes. Furthermore, when a similar analysis was performed on unbound molecules present in the free cytoplasm, identical results were obtained. We conclude that no selection towards poly(rA)-containing viral molecules is evident on binding to polyribosomes. These findings suggest that the incoming viral genome of the Moloney strain of murine leukemia virus may serve as a messenger for the synthesis of one or more virus-specific proteins early after infection of mouse fibroblasts.     

Translation of enzymically decapped messenger RNA.

An enzymic procedure was used to remove the 7-methylguanosine diphosphate moiety at the 5' ends of rabbit hemoglobin mRNA and mouse immunoglobulin light-chain mRNA. Evidence was obtained that the procedure, which involves the use of polynucleotide kinase, does not result in any further degradation of the mRNA. The enzymically decapped mRNA was as effective as untreated mRNA in supporting protein synthesis in a wheat germ system. This was the case over a wide range of mRNA concentrations and over a considerable period of time. The presence in the incubation mixture of S-adenosylhomocysteine, an inhibitor of methylation, did not affect the results. The data indicate that the presence of a 7-methylguanosine diphosphate residue at the 5' end of mRNAs is not an obligatory requirement for translation in eucaryotic systems.