Antisense RNAs as envoys in intercellular communication: 20 years later.

More than 20 years ago we showed that some types of cells are capable of secreting RNAs. It was suggested that these secreted RNAs could serve as molecular envoys in intercellular communication, for example, these RNAs being complementary to specific sites of the gene in another cell (e.g. to the variable region of immunoglobulin gene) could regulate the expression of genes that contain sites in coding regions complementary to antisense RNA. It has since been proven that eukaryotic cells contain antisense RNAs (particularly microRNAs and small interfering RNAs), which can regulate the expression of genes at the posttranscriptional level (the so-called regulatory pathway of RNA interference). Here I provide a short review of advances in the field of intracellular regulation of gene expression by different types of RNAs. In addition, an overview of recent data on the secretion of RNA molecules by different cell types and possible involvement of these secreted antisense RNAs in intercellular regulation of gene expression in target cells is given.

The phenotype of mutations of the base-pair C2658.G2663 that closes the tetraloop in the sarcin/ricin domain of Escherichia coli 23 S ribosomal RNA.

The sarcin/ricin domain (SRD) in Escherichia coli 23 S rRNA is a part of the site for the association of elongation factors with ribosomes and for that reason is critical for the binding of aminoacyl-tRNA and for translocation during the reiterative elongation reactions of protein synthesis. The SRD has a GAGA tetraloop that is shut off by a Watson-Crick C2658 x G2663 pair. The contribution of this pair to the function of the ribosome has been evaluated by constructing mutations in the nucleotides and determining their phenotype. Constitutive expression of a plasmid-encoded rrnB operon with a G2663C transversion mutation that disrupts the Watson-Crick pair was lethal. Double transversion mutations, C2658G x G2663C and C2658A x G2663U, that reverse the polarity of the pyrimidine and the purine but restore the potential to form a canonical pair, were also lethal. Induction of transcription of 23 S rRNA with the same mutations, but encoded in a plasmid with a lambdaP(L) promoter and expressed at a lower level, retarded growth. The sedimentation profiles of ribosomes with transversion mutations in C2658 and/or G2663 are altered; the ratio of 50 S subunits to 30 S particles is changed and polysomes are reduced. Ribosomes with a G2663C, a C2658G x G2663C, or a C2658A x G2663U mutation in 23 S rRNA were not active in protein synthesis, indeed, they appeared to inhibit the activity of ribosomes with wild-type 23 S rRNA. Transversion mutations in the analogs of C2658 and G2663 decreased binding of EF-G to SRD oligoribonucleotides; the same mutations in 23 S rRNA decreased binding of the factor to intact ribosomes. The most severe phenotype, in growth, in protein synthesis, and in the binding of EF-G, was associated with a C2658G x G2663C mutation; it is surprising that this was more severe than an analogous C2658A x G2663U mutation. A double transition mutation, C2658U x G2663A, which is not known to have occurred in nature, had no effect on the growth of cells or on the function of ribosomes. The lethal phenotype of transversion mutations in C2658 and G2663 appears to derive from a loss of the capacity of ribosomes to bind EF-G and by indirection the EF-Tu ternary complex.

Preferential interaction of the mRNA proofreading factor TFIIS zinc ribbon with rU.dA base pairs correlates with its function.

The transcriptional factor TFIIS helps overcome elongation barriers and enhances proofreading by RNA polymerase II. These TFIIS functions may be modulated by the TFIIS zinc ribbon domain through interactions with nucleic acids in the elongation complex. Within this zinc ribbon domain, the dipeptide sequences Asp261-Glu262 and Arg276-Trp277 have been shown to be critical for its function by mutant analysis. The sequence Asp261-Glu262 has been suggested to participate in metal binding within the RNA polymerase II active site. We now show that the sequence Arg276-Trp277 interacts with nucleic acids through a combination of electrostatic and stacking interactions. The interaction of the indole side chain of the tryptophan residue with nucleic acid bases is demonstrated by a characteristic and reversible decrease in the zinc ribbon fluorescence intensity as a function of oligonucleotide concentration. These interactions are salt sensitive (maximum interaction at 200 mM and no interaction at 500 mM NaCl), suggesting that the tryptophan stacking with nucleic acid base accompanies electrostatic contacts. The oligonucleotide-zinc ribbon interactions exhibit small but significant base preferences, as shown by the dependence of Keq on base composition, with decreasing Keq in the order U > T > A > C >> G. Within the variety of homopolymeric single- and double-stranded deoxy- and ribooligonucleotides, the oligonucleotide rU12-18.dA20 exhibited a 2-6-fold binding preference relative to other oligonucleotides. This preferential binding of the zinc ribbon to sequences composed of rU.dA base pairs, which are generally associated with elongation blocks, may help in overcoming elongation barriers. Since the mRNA proofreading and enhancement of elongation involve cleavage of ribonucleotide of the mismatched pair and the weakly paired rU.dA nucleotides, but not the stably paired rC.dG nucleotides, we propose that the Arg276-Trp277 sequence in the TFIIS zinc ribbon may serve as a scanner connected to the transcript cleavage apparatus for weakly paired or mismatched nucleotides by employing indole ring stacking with the bases as a criterion of determining their subsequent removal. The striking similarity in preference for mismatched and weakly paired nucleotides for binding and for excision suggests a functional relationship between binding and cleavage reactions.

The major protein of messenger ribonucleoprotein particles in somatic cells is a member of the Y-box binding transcription factor family.

A cDNA encoding the major core protein, p50, of cytoplasmic messenger ribonucleoprotein particles (mRNPs) of somatic cells was cloned from a rabbit reticulocyte cDNA library. From the derived 324-amino acid sequence, p50 is identified as a member of the Y-box binding transcription factor family. The protein was earlier described as a repressor of globin mRNA translation. These findings suggest that p50 may affect protein biosynthesis at two levels: mRNA transcription in the nucleus and mRNA translation in the cytoplasm. Together with recently published results showing that masked mRNA in germ cells also is associated with proteins of the Y-box binding protein family, the present finding indicates that these proteins are universal core proteins responsible for the formation of cytoplasmic mRNPs in eukaryotes. Highly purified p50 forms large 18 S homomultimeric complexes with a molecular mass of about 800 kilodaltons and melts RNA secondary structure. This suggests that p50 may affect translation by changing the overall structure of the mRNA.

cAMP-dependent activation of protein synthesis correlates with dephosphorylation of elongation factor 2.

The addition of 5 mM cAMP to a cell-free translation system from rabbit reticulocytes increases the rate of protein synthesis 3 5-fold. Lower concentrations of cAMP (0.005, 0.05 and 0.5 mM) have no effect on translation in this system. cAMP at all the concentrations tested stimulates the phosphorylation of the same pattern of polypeptides, while 5 mM cAMP additionally stimulates dephosphorylation of the 95 kDa polypeptide identified as elongation factor 2 (EF-2). Testing of the preparations of EF-2 with a different content of the phosphorylated form in poly(U)-directed poly(Phe) synthesis reveals that the EF-2 activity correlates with the fraction of non-phosphorylated EF-2. Thus cAMP-dependent activation of protein synthesis seems to be due to dephosphorylation of EF-2.

Endogenous ADP-ribosylation of elongation factor 2 in polyribosome fraction of rabbit reticulocytes.

Several polypeptides of about 120, 96, 85, 60 and 38 kDa are shown to be radiolabeled during incubation of the mono- and polyribosome fraction of rabbit reticulocytes with [32P]NAD. Among them is a polypeptide coinciding with elongation factor 2 (EF-2) in its electrophoretic mobility in SDS-polyacrylamide gel. The addition of pure EF-2 to the polyribosome fraction results in an increase of the radioactive label in this polypeptide band. From this it is concluded that both endogenous and added EF-2 is ADP-ribosylated by an enzyme associated with polyribosomes. A possibility of regulation of protein synthesis through endogenous ADP-ribosylation in vivo is considered.

Eukaryotic elongation factor 2 loses its non-specific affinity for RNA and leaves polyribosomes as a result of ADP-ribosylation.

ADP-ribosylation of rabbit reticulocyte elongation factor 2 (EF-2) catalyzed by the A fragment of diphtheria toxin leads to a loss of its non-specific affinity for RNA. The removal of the ADP-ribose residue from EF-2 in the reverse reaction with nicotinamide restores its affinity for RNA. ADP-ribosylation of EF-2 is accompanied by its dissociation from the complexes with mono- and polyribosomes detected in the rabbit reticulocyte lysate at low ionic strength. The loss of the non-specific affinity of EF-2 for RNA as a result of ADP-ribosylation and, as a consequence, its decompartmentation from polyribosomes is assumed to be a reason for the diphtheria toxin-induced inactivation of the factor in eukaryotic cells.

Phosphorylation of elongation factor 1 in polyribosome fraction of rabbit reticulocytes.

A single protein, Mr approximately 50000, is shown to be phosphorylated during incubation of a mono- and polyribosome fraction of rabbit reticulocytes with [gamma-32P]ATP at a low ionic strength. This protein has been identified as the elongation factor 1 alpha (EF-1 alpha). The phosphorylated EF-1 alpha, in contrast to the unmodified factor, is not detected in complexes with mono- and polyribosomes. It is suggested that the phosphorylation of EF-1 alpha can result in its decompartmentation from polyribosomes and thus affect the rate of protein synthesis.