Messenger RNA degradation: beginning at the end

The mechanisms responsible for mRNA decay in mammalian cells, and how specific sequence elements accelerate decay, are unknown. Recent work indicates that 'ARE' instability elements recruit the exosome to promote rapid 3'-to-5' degradation of the mRNA.     

Sequence of 3000 nucleotides at the 5' end of Japanese encephalitis virus RNA

The 5' region of the Japanese encephalitis virus (JEV) RNA was cloned and 3000 nucleotides (nt) were determined by sequencing DNA complementary to viral RNA, and genomic RNA, using oligodeoxynucleotide primers and the dideoxy chain-termination reaction. Comparison of the nt sequence and the reduced amino-acid sequence of JEV with those of other flaviviruses showed significant homologies, which allowed locations to be assigned for three structural proteins.     

Evidence that the 5' end of rabbit globin mRNA is hybridized with its 3' end

• 1.1. A radiopolyadenylated rabbit globin mRNA was treated with different concentrations of ribonuclease V₁ from cobra venom.
• 2.2. The enzymatic digests were chromatographed on an aminophenylboronate-agarose column, which specifically captured the cap structure i.e. n⁷G(5') ppp (5') NmP.
• 3.3. When the capture fragment was chromatographed on a Sephadex G-100 column, its size was smaller than the native molecule and also bore radioactivity, i.e. a poly(A) tail.
• 4.4. These results provide evidence that the 5' end (which encompasses the cap structure) of rabbit globin mRNA is hybridized and in close proximity to its 3' end.
• 5.5. We conclude that this conformation is required for messenger translation efficiency.

     

Origin and degradation of the RNA primers at the 5' termini of nascent DNA chains in Bacillus subtilis

We had earlier characterized the nascent DNA synthesized in permeable cells of Bacillus subtilis in the presence of 5-mercurideoxycytidine triphosphate and 2',3'-dideoxyATP as being substituted at its 5' end with a ribonucleotide moiety of the sequence pApG(pC)1-2 DNA. In this paper, we examine the origin and turnover of the DNA-linked ribonucleotide and its relationship to DNA replication. At least 50% of the RNA-linked nascent DNA chains served as guanylate acceptors when incubated with GTP and the eukaryotic capping enzyme, indicating the presence of 5'-terminal di- or triphosphate groups and suggesting that the RNA moiety is synthesized de novo and is not a degradation product. In nascent DNA produced without limitation of chain growth by dideoxyATP, the degree of terminal ribonucleotide substitution was reduced by 50%, consistent with a linkage between RNA primer removal and DNA chain growth. Such a relationship was demonstrated directly by examining the RNA primer content of nascent DNA synthesized in the absence of dideoxyATP as a function of DNA chain length. As the DNA size increased from 40 to 200 nucleotide residues, the extent of RNA substitution declined from 80% to nearly 0%. Endgroup analysis showed that the loss of RNA was accompanied by a gradual shift from predominantly adenylate residues to 5'-terminal guanylate, consistent with a stepwise removal of ribonucleotides from the 5' end. Evidence that the nascent mercurated DNA synthesized under our experimental conditions was indeed a replicative intermediate came from the study of the time course of DNA chain growth and pulse-chase experiments. In the presence of the DNA ligase inhibitor NMN, mercurated DNA accumulated in two size classes with average length of approximately 750 and 8000 nucleotide residues, presumably representing the mature size of intermediates in discontinuous DNA synthesis. Comparison with the DNA size range at which the loss of the 5'-terminal RNA moiety occurred (40 to 200 residues) indicated that the processing of RNA primers occurred at an early stage during DNA chain elongation, and that moderate size intermediates in discontinuous DNA replication (greater than 200 nucleotides) have already lost their RNA primers.     

Protein is linked to the 5' end of poliovirus RNA by a phosphodiester linkage to tyrosine.

Purification and partial characterization of the poliovirus RNA-linked protein (VPg) are described. VPg has been freed from the RNA by ribonuclease digestion and phenol extraction. Gel filtration chromatography of VPg-pUp (labeled with 32P) in 0.5% sodium dodecyl sulfate or 6 M guanidine HCl indicates that it has a molecular weight of about 12,000. VPg is bound to the 5' end of poliovirion RNA by a phosphodiester bond between a tyrosine residue in the VPg molecule and the 5'-terminal uridine. After acid hydrolysis of [3H]tyrosine-labeled VPg-pU, free tyrosine can be released by venom phosphodiesterase. Acid hydrolysis of VPg-p labeled with either 32P or [3H] tyrosine yields tyrosine-phosphate. There appears to be only 1 tyrosine residue per VPg molecule.     

Structural characterization of the highly conserved 98-base sequence at the 3' end of HCV RNA genome and the complementary sequence located at the 5' end of the replicative viral strand

Oligoribonucleotides that corresponded to the X regions of the (+) and (−) polarity strands of HCV RNA, as well as several shorter oligomers comprising defined stem-loop motifs of their predicted secondary structure models, were analyzed by Pb²⁺-induced cleavage, partial digestion with specific nucleases and chemical modification. Patterns characteristic of the motifs were compared with those obtained for the full-length molecules and on the basis of such ‘structural fingerprinting’ conclusions concerning folding of regions X were formulated. It turned out that the secondary structure model of X(+) RNA proposed earlier, the three-stem-loop model composed of hairpins SL1, SL2 and SL3, was only partially consistent with our experimental data. We confirmed the presence of SL1 and SL3 motifs and showed that the single-stranded stretch adjacent to the earlier proposed hairpin SL2 contributed to the folding of that region. It seemed to be arranged into two hairpins, which might form a hypothetical pseudoknot by changing their base-pairing systems. These data were discussed in terms of their possible biological significance. On the other hand, analysis of the X(−) RNA and its sub-fragments supported a three-stem-loop secondary structure model for this RNA.     

A functional ribonucleoprotein complex forms around the 5' end of poliovirus RNA

The existence of a computer-predicted cloverleaf structure for the first 100 nucleotides at the 5' end of poliovirus RNA was verified by site-directed mutagenesis and by chemical and RNAase probing. Mutations that modified the cloverleaf in the positive strand but not the negative strand were lethal to the virus. This RNA cloverleaf structure binds a cellular protein and the viral proteins 3Cpro and 3Dpol. Mutations in specific regions of the RNA cloverleaf prevented this binding. Mutations in either 3Cpro or the RNA that disrupted ribonucleoprotein complex formation inhibited virus growth and selectively affected positive strand RNA accumulation. Phenotypic reversion of these mutations restored the ability to form the complex. Thus, a cloverleaf structure in poliovirus RNA plays a central role in organizing viral and cellular proteins involved in positive strand production.