Poly (A) sequences in insect RNA

RNA isolated from the epidermis and fat body of larvae of the insect Calliphora vicina, contains poly (A) sequences, as demonstrated by binding to nitrocellulose filters and to oligo-dT cellulose. The RNA eluted from the oligo-dT cellulose columns was treated with DNAase, pancreatic RNAase and T₁ nuclease. The size of the nuclease resistant polynucleotides was determined by acrylamide gel electrophoresis and by alkaline hydrolysis and measurement of the adenylic acid to adenosine ratio, the former method yielding 120 to 140 the latter 50 to 65 adenylic acid residues.     

Poly(A)-associated RNA in plants

The RNA associated with poly(A) sequences from Euglena gracilis and Vicia faba has been isolated by binding to millipore filters and characterized by sedimentation velocity centrifugation and electrophoretic mobility. Poly(A)-associated RNA as isolated in solution was highly aggregated. When denatured, it sedimented as a broad peak with a mean value of 16-18 S. This RNA was shown to be covalently linked to poly(A) sequences which are 150-250 nucleotides long. Our size estimates for plant poly(A) and poly(A)-associated RNA are similar to those obtained for animal cells.     

Poly(A) on mengovirus RNA.

The content and size of the poly(A) on Mengovirus RNA grown in both mouse L cells and HeLa cells have been examined. Virion RNA from either cell line could bind to poly(U) filters and contained RNase-resistant stretches of poly(A) which could be analyzed by electrophoresis in polyacrylamide gels. The size of the poly(A) on the Mengovirus RNA was independent of the host cell and averaged from 50 to 70 nucleotides.     

Poly(A) RNA and Paip2 act as allosteric regulators of poly(A)-binding protein

When bound to the 3′ poly(A) tail of mRNA, poly(A)-binding protein (PABP) modulates mRNA translation and stability through its association with various proteins. By visualizing individual PABP molecules in real time, we found that PABP, containing four RNA recognition motifs (RRMs), adopts a conformation on poly(A) binding in which RRM1 is in proximity to RRM4. This conformational change is due to the bending of the region between RRM2 and RRM3. PABP-interacting protein 2 actively disrupts the bent structure of PABP to the extended structure, resulting in the inhibition of PABP-poly(A) binding. These results suggest that the changes in the configuration of PABP induced by interactions with various effector molecules, such as poly(A) and PABP-interacting protein 2, play pivotal roles in its function.     

Poly(A) sequences in Naegleria messenger RNA before and after initiation of differentiation : Absence of oligo(A)

The ameboid stage of the amebo-flagellate Naegleria gruberi was found to synthesize two size classes of polynucleotides resistant to digestion with a mixture of ribonuclease A and T1. These two size classes were present in both the nucleus and the cytoplasm. Cells differentiating into flagellates were found to lose a variable amount of the smaller, nuclease-resistant fragment while synthesizing only the larger nuclease-resistant class. The adenosine to AMP ratio of the larger nuclease-resistant fragment was compatible with a 3′-terminal poly(A) sequence of 87 nucleotides average length. The smaller nuclease-resistant fragment was found to be rich in AMP (44–49%) but contained a substantial amount of other nucleotides. The smaller fragment was heterogeneous in size with an average length of 10–12 nucleotides as estimated by its elution from a DEAE column. Fractionation of RNA on oligo(dT) cellulose demonstrated that the large and small nuclease-resistant fragments were on different RNA molecules. Only the large poly(A) sequence was present in either cytoplasmic or nuclear RNA which bound to oligo(dT) cellulose. On the other hand, only the small nuclease resistant fragment was found in the unbound RNA from either nuclei or cytoplasm.     

Poly(A)-containing RNA from germinating wheat embryos

Rapidly labelled mRNAs were isolated from informosomes and polyribosomes of imbibed wheat embryos. The distribution of poly(A) sequences in these fractions were studied by poly(U) Sepharose chromatography. It was shown that informosomes contain 11% polyadenylated mRNA while polyribosomes--38%. This fact suggests the important role of poly(A) sequences for translation of mRNA.     

Poly(A)-containing RNA in the livers of ethionine-treated rats

Ethionine intoxication in nonfasted rats leads to a rapid change in the levels of poly(A)+ RNA. Prolonged fasting causes a decrease in poly(A)+ RNA which is not aggravated further by ethionine. The size distribution of poly(A) tracts at the 3' end of mRNA is not affected by ethionine. However, ethionine induces a change in the metabolism of the poly(A) sequence relative to the remainder of the mRNA.     

Poly(A)-containing RNA in early embryogenesis of sea urchins

The content, biosynthesis and template activity of poly(A)+ RNA in the early stages of sea urchin development have been studied. The amount of poly(A)+ RNA reaches a maximum at the middle blastula stage in polyribosomes and at the 8-blastomere stage in the cytoplasm. Poly(A)+ RNA synthesis becomes noticeable at the 64-blastomere stage and the spectrum of newly synthesized molecules is different from that at the middle blastula stage. The products of translation in vitro of poly(A)+ RNA at all the stages studied show insignificant differences and contain a major group of polypeptides of molecular mass 10-20 kDa.     

Evidence that all messenger RNA molecules (except histone messenger RNA) contain Poly (A) sequences and that the Poly(A) has a nuclear function

The appearance of newly formed messenger RNA in polyribosomes of HeLa cells Is inhibited by over 85% by 3′deoxyadenosine (Penman, Rosbash &; Penman, 1970) probably due to the failure of normal attachment of poly(A) to heterogeneous nuclear RNA in the presence of this drug (Darnell, Philipson, Wall &; Adesnik, 1971). Results presented here show that the labeled RNA which does reach polysomes in the presence of 3′deoxyadenosine can be characterized as messenger RNA which contains smaller poly(A) segments than normal messenger RNA. The results of the present experiments suggest that all, or almost all, HeLa cell messenger RNA molecules (except for histone messenger RNA) are derived from nuclear RNA molecules which contain poly (A).     

Poly(A)- and primer-independent RNA polymerase of Norovirus

Replication of positive-strand caliciviruses is mediated by a virus-encoded RNA-dependent RNA polymerase (RdRp). To study the replication of Norovirus (NV), a member of the family Caliciviridae, we used a recombinant baculovirus system to express an enzymatically active RdRp protein from the 3D region of the NV genome and defined conditions for optimum enzymatic activity. Using an RNA template from the NV 3' genomic region, we observed similar levels of enzymatic activity in assays with and without a poly(A) tail. RdRp activity was not significantly affected by the addition of an RNA primer to the reaction mixture. Thus, the NV RdRp exhibited primer- and poly(A)-independent RNA polymerase activity. While the RdRp inhibitor phosphonoacetic acid inhibited NV RdRp activity, another gliotoxin did not. The active recombinant NV RdRp will be of benefit to studies of NV replication and will facilitate the development of specific inhibitors of NV proliferation.     

Properties and Location of Poly(A) in Rous Sarcoma Virus RNA

The poly(A) sequence of 30 to 40S Rous sarcoma virus RNA, prepared by digestion of the RNA with RNase T(1), showed a rather homogenous electrophoretic distribution in formamide-polyacrylamide gels. Its size was estimated to be about 200 AMP residues. The poly(A) appears to be located at or near the 3' end of the 30 to 40S RNA because: (i) it contained one adenosine per 180 AMP residues, and because (ii) incubation of 30 to 40S RNA with bacterial RNase H in the presence of poly(dT) removed its poly(A) without significantly affecting its hydrodynamic or electrophoretic properties in denaturing solvents. The viral 60 to 70S RNA complex was found to consist of 30 to 40S subunits both with (65%) and without (approximately 30%) poly(A). The heteropolymeric sequences of these two species of 30 to 40S subunits have the same RNase T(1)-resistant oligonucleotide composition. Some, perhaps all, RNase T(1)-resistant oligonucleotides of 30 to 40S Rous sarcoma virus RNA appear to have a unique location relative to the poly(A) sequence, because the complexity of poly(A)-tagged fragments of 30 to 40S RNA decreased with decreasing size of the fragment. Two RNase T(1)-resistant oligonucleotides which distinguish sarcoma virus Prague B RNA from that of a transformation-defective deletion mutant of the same virus appear to be associated with an 11S poly(A)-tagged fragment of Prague B RNA. Thus RNA sequences concerned with cell transformation seem to be located within 5 to 10% of the 3' terminus of Prague B RNA.     

Poliovirus Infection and Poly(A) Sequences of Cytoplasmic Cellular RNA

The influence of polio infection on Poly(A) sequences of cellular cytoplasmic RNA was investigated. In the presence of guanidine, cellular protein synthesis was still shut off after poliovirus infection, although there was no viral RNA synthesis. The Poly(A) of these cells was unchanged with respect to quantity, size, and linkage to cellular cytoplasmic RNA. This finding strongly suggests that the shut off of cellular protein synthesis is not caused by a change of the Poly(A) sequences of cellular mRNA.     

Poly(A) synthesis by RNA polymerase II from rat liver

Multiple forms of DNA-dependent RNA polymerase were resolved by DEAE-Sephadex chromatography. In addition to RNA polymerases, an active poly(A) polymerase was also fractionated. RNA polymerases were examined for their capacity to synthesize poly(A). None of the freshly prepared enzymes could efficiently make poly(A) in presence or absence of exogenous primers. However, “aging” of polymerase II by simple incubation at 37°C resulted in the loss of RNA polymerizing activity with a corresponding increase in poly(A) synthesizing activity. Transformation of RNA polymerase to poly(A) polymerase resulted in reduced capacity to transcribe native DNA and altered chromatographic behavior. The results suggest that subunits of polymerase II obligatory to DNA-dependent RNA synthesis were degraded by “aging” and that a stable subunit of the RNA polymerase could preferentially make poly(A).     

Poly(A) and synthesis of polyadenylated RNA in the preimplantation mouse embryo

Investigations were conducted to quantitate polyadenylic acid and estimate the synthesis of polyadenylated RNA in mouse embryos at several stages of preimplantation development. Poly(A) was assayed by molecular hybridization of total embryonic RNA with [³H]polyuridylic acid. The mean values of poly(A) in the ovulated oocytes and in the one-cell, two-cell, and blastocyst stages of the embryo were 1.9, 1.6, 0.68, and 3.8 pg, respectively. Synthesis of polyadenylated RNA was estimated by affinity chromatography of [³H]uridine-labeled embryo RNA on oligo(dT)-cellulose. The proportions of newly synthesized RNA bound by oligo(dT)-cellulose at the 2-cell, 8- to 16-cell, and blastocyst stages were 6.7, 3.5, and 3.3%, respectively. These results suggest that significant quantities of maternal mRNA are present during early development of the mouse, but that polyadenylation of RNA transcribed from the embryonic genome occurs as early as the two-cell stage.