Induction of 2',5' oligo(A) synthetase in tumor-bearing mice with encephalomyocarditis (EMC) virus or poly(I)poly(C)

Infection of 13 month-old C3H mice with EMC virus or inoculation with the interferon inducer poly(I)poly(C) results in elevated levels of the enzyme 2',5' oligo(A) synthetase only in animals with spontaneous tumors (breast cancer or hepatomas). High enzymatic activities are detected in homogenates from liver, spleen, plasma and neoplastic cells of the animals with breast carcinomas and only in the neoplastic liver cells of the animals with hepatomas.     

Presence of 2',5'-oligo(A) and of enzymes that synthesize, bind, and degrade 2',5'-oligo(A) in HeLa cell nuclei

Nuclei prepared from HeLa cells by lysis with nonionic detergents or by a nonaqueous fractionation procedure were assayed for enzymatic activities which synthesize, bind, and degrade 2',5'-oligo(A). Isolated nuclei synthesized micromolar concentrations of 2',5'-oligo(A) when incubated with poly(inosinic) . poly(cytidylic) acid. The products of nuclear synthesis were identified with authentic 2',5'-oligo(A) by several criteria. The nuclei synthesized nanomolar amounts of 2',5'-oligo(A) even when incubated without added double-stranded RNA. These oligonucleotides were identified by their pattern of degradation with different nucleases and by a specific competition-binding assay. This assay revealed the presence in nuclei of an activity which binds 2',5'-oligo(A) with an affinity constant similar to that of the cytoplasmic binding activity previously identified with the 2',5'-oligo(A)-dependent endoribonuclease (Nilsen, T. W., Wood, D. L., and Baglioni, C. (1981) J. Biol. Chem. 256, 10751-10754). The nuclei had also an activity which degraded 2',5'-oligo(A). Finally, unincubated nuclei isolated by the nonaqueous fractionation procedure contained detectable concentrations of 2',5'-oligo(A). These results show that an activator of the enzyme which synthesize 2',5'-oligo(A) is present in nuclei and that these oligonucleotides are normally formed in HeLa cells, and suggest a possible role for the 2',5'-oligo(A)-activated endoribonuclease in nuclear RNA metabolism.     

The Elav-like proteins bind to AU-rich elements and to the poly(A) tail of mRNA.

The Elav-like proteins are specific mRNA binding proteins which are required for cellular differentiation. They contain three characteristic RNP2/RNP1-type RNA binding motifs. Previously we have shown that the first and second RNA binding domains bind to AU-rich elements in the 3'-UTR of mRNA. In this paper we show that the Elav-like proteins exhibit poly(A) binding activity. This activity is distinct from poly(A) binding activities that have been previously described. The Elav-like proteins specifically bind to long chain poly(A) tails. We have shown that the third RNA binding domain encompasses this poly(A) binding activity. Using poly(A)-Sepharose beads in a 'sandwich' assay we have shown that the Elav-like proteins can bind simultaneously to the AU-rich element and to the poly(A) tail.     

Diphtheria toxin can simultaneously bind to its receptor and adenylyl-(3',5')-uridine 3'-monophosphate

Diphtheria toxin that was bound to receptors on BS-C-1 cells was able to bind approximately 1 molar equiv of adenylyl-(3',5')-uridine 3'-monophosphate (ApUp). In contrast, receptor-bound CRM197, a mutant form of toxin with greatly diminished affinity for dinucleotides, did not bind ApUp. Affinity of the dinucleotide for receptor-bound toxin differed from that for free toxin by less than an order of magnitude. These results indicate that the receptor site and the ApUp site on the toxin do not significantly overlap.     

Methylated pyrimidines stabilize an alternating conformation of poly(dA-dU).poly(dA-dU)

We have investigated the effect of increasing percentages of methylated pyrimidines on the structure of poly(dA-dU).poly(dA-dU). This was done by synthesizing analogous polynucleotides that contained deoxythymidine residues as well as deoxyuridine residues and observing their 31P NMR spectra in increasing amounts of CsF. The results show that methylated pyrimidines play a large role in the stabilization of the "alternating B" conformation of DNA.     

Spermine inhibition of the 2,5-diaziridinyl-1,4-benzoquinone (DZQ) crosslinking reaction with DNA duplexes containing poly(purine). poly(pyrimidine) tracts.

Upon reduction, 2,5-diaziridinyl-1,4-benzoquinone (DZQ) can form an interstrand guanine to guanine crosslink with DNA duplexes containing a d(GC).d(GC) dinucleotide step. The reaction is enhanced by a thymine positioned 5[prime] to each guanine [i.e. in a d(TGCA). d(TGCA) duplex fragment]. Here we show that spermine can inhibit DZQ crosslink formation in duplexes of sequence d[C(N6)TGCA(M6)C]. d[G(M[prime]6)TG-CA(N[prime]6)G]. For N6= M6= GGGGGG, N6= M6= a 'random' sequence and N6= GGGGGG and M6= a 'random' sequence, spermine concentrations of 20, 1 and 3 microM, respectively, were required for 50% inhibition of the DZQ crosslink. This suggests that spermine is more strongly bound to the polyguanosine tract than the random sequence, making it less available for crosslink inhibition. When the polyguanosine tract is interrupted by N 7-deazaguanine (D) located three bases, d(CGGGDGGTGCAGGDGGGC), and four bases, d(CG-GDGGGTGCAGGGDGGC), from the d(TGCA).d(TGCA) site, 30 and 3 microM spermine, respectively, were required for 50% crosslink inhibition. We suggest that this difference is due to the relative proximity of the three-guanosine tract to the d(TGCA).d(TGCA) site. We were able to confirm these conclusions with further experiments using duplexes containing three-guanosine and two-guanosine tracts and from computer simulations of the spermine-DNA complexes.     

The 5' and 3' domains of yeast U6 snRNA: Lsm proteins facilitate binding of Prp24 protein to the U6 telestem region

The 5' and 3' domains of yeast U6 snRNA contain sequences that are thought to be important for binding to Prp24 and Lsm proteins. By extensive mutational analysis of yeast U6 snRNA, we confirmed that the 3' terminal uridine tract of U6 snRNA is important for U6 binding to Lsm proteins in yeast. Binding of Prp24 protein to U6 RNA is dependent on or is strongly enhanced by U6 binding of Lsm proteins. This supports a model for U6 snRNP assembly in which U6 RNA binds to the Lsm2-8 core prior to binding Prp24 protein. Using compensatory base-pairing analysis, we show that at least half of the recently identified U6 telestem as well as a nucleotide sequence in the other half of the telestem are important for binding of U6 RNA to Prp24 protein. Surprisingly, disruption of base pairing in the unconfirmed half of the telestem enhanced U6-Prp24 binding. Truncation of the entire 3' terminal domain or nearly the entire 5' terminal domain of yeast U6 allowed for detectable levels of splicing to proceed in vitro. In addition to gaining knowledge of the function of the 5' and 3' domains of yeast U6, our results help define the minimal set of requirements for yeast U6 RNA function in splicing. We present a revised secondary structural model of yeast U6 snRNA in free U6 snRNPs.     

A convenient synthesis of adenylyl-(2'----5')-adenylyl-(2'----5')-adenosine (2-5A core)

A simple synthesis of adenylyl-(2'----5')-adenylyl (2'----5')-adenosine (2-5A core) has been achieved on the basis of selective 3'-O-silylation of 5'-O-p-monomethoxytrityladenosine and chemo-selective formation of the 2'-5' internucleotide linkage using N-unprotected nucleosides.     

Structure and function of poly(A) binding proteins

Poly (A) tails are found at the 3' ends of almost all eukaryotic mRNAs. They are bound by two different poly (A) binding proteins, PABPC in the cytoplasm and PABPN1 in the nucleus. PABPC functions in the initiation of translation and in the regulation of mRNA decay. In both functions, an interaction with the m7G cap at the 5' end of the message plays an important role. PABPN1 is involved in the synthesis of poly (A) tails, increasing the processivity of poly (A) polymerase and contributing to defining the length of a newly synthesized poly (A) tail.