Regulated immunoglobulin (Ig) RNA processing does not require specific cis-acting sequences: non-Ig RNA can be alternatively processed in B cells and plasma cells.

Alternative RNA processing of the heavy-chain immunoglobulin mu gene is regulated during B-cell maturation and requires competition between splice and cleavage-polyadenylation reactions that have balanced efficiencies. Studies with modified mu genes have failed to identify gene-specific sequences required for regulation. Thus, the only important feature for regulation may be the balanced competing splice and cleavage-polyadenylation reactions themselves. If this is so, then alternative RNA processing from any gene with similar competitive RNA processing pathways should also be regulated when expression is compared between B cells and plasma cells. To test this prediction, two nonimmunoglobulin genes engineered to have competing splice and cleavage-polyadenylation reactions were expressed in B cells and plasma cells. The ratios of alternative RNAs produced from both genes are different in the two cell types; like the mu gene, relatively more spliced RNA is produced in B cells than in plasma cells. Also, in a survey of mu gene expression in nine non-B-cell lines, only a T-cell line had an expression pattern similar to that of B cells; the expression patterns of all other lines resembled that of the plasma cells. Therefore, regulated mu RNA processing must be mediated by changes in general processing factors whose activity or abundance is regulated, most likely, in B cells.     

Determination of relative rate constants for in vitro RNA processing reactions by internal competition

Studies of RNA recognition and catalysis typically involve measurement of rate constants for reactions of individual RNA sequence variants by fitting changes in substrate or product concentration to exponential or linear functions. A complementary approach is determination of relative rate constants by internal competition, which involves quantifying the time-dependent changes in substrate or product ratios in reactions containing multiple substrates. Here, we review approaches for determining relative rate constants by analysis of both substrate and product ratios and illustrate their application using the in vitro processing of precursor transfer RNA (tRNA) by ribonuclease P as a model system. The presence of inactive substrate populations is a common complicating factor in analysis of reactions involving RNA substrates, and approaches for quantitative correction of observed rate constants for these effects are illustrated. These results, together with recent applications in the literature, indicate that internal competition offers an alternate method for analyzing RNA processing kinetics using standard molecular biology methods that directly quantifies substrate specificity and may be extended to a range of applications.     

RNA METABOLISM IN HELA CELLS AT REDUCED TEMPERATURE : I. Modified Processing of 45S RNA

Incubation of HeLa cells at suboptimal temperature has been used to study the synthesis of 45S ribosomal RNA precursor and the individual steps of the subsequent processing to 28S RNA. Below 20°C no detectable 45S RNA is formed. The processing of 45S RNA to 32S RNA ceases around 15°C, and the processing of 32S RNA to 28S RNA is inhibited near 25°C. Prolonged incubation at reduced temperature results in further modification of the processing, resulting in the apparent accumulation of 41S RNA. The products of these reactions at reduced temperature appear normal in that the ribosomal RNA made at 27°C can be isolated from functional polyribosomes in the cytoplasm after a short incubation at 37°C.     

Two autolytic processing reactions of a satellite RNA proceed with inversion of configuration.

Both polarities of the satellite RNA of tobacco ringspot virus occur in infected cells in multimeric forms which are capable of autolytic processing, using different sequences and structures [Feldstein, P.A., et al., Proc. Nat. Acad. Sci. USA (1990) 87 (in press)]. These transesterification reactions generate a 2',3'-cyclophosphate and a 5'-hydroxyl as the two new end groups. Cleavage is at a CpA for the (+) polarity RNA and at an ApG for the (-) polarity RNA. We enzymically synthesized oligoribonucleotides with processing capability and with specific 35S-labeled phosphorothioate diesters in the Rp configuration. After processing had occurred, the terminal nucleoside-2',3'-cyclophosphorothioate diester residues were recovered from the appropriate product by digestion with nuclease and phosphatase. Comparisons with specially prepared endo- and exoisomer reference compounds by thin layer chromatography and autoradiography revealed that the [35S]cytidine- and [35S]adenosine-2',3'-cyclophosphorothioate both were endo-isomers. The results are consistent with transesterification occurring by an inline SN2(P) attack of the 2'-hydroxyl group in the autolytic processing reactions of both polarities of the satellite RNA.     

RNA mediated formation of a phosphorothioate diester bond

Previous results showed that multimeric, tandemly sequence-repeated forms of satellite tobacco ringspot virus RNA of the encapsidated polarity (STobRV (+)RNA) autolytically process at a specific phosphodiester bond, the junction. Substituting a phosphorothioate diester bond for the STobRV (+)RNA junction drastically slowed autolytic processing. Here we show that for the complementary STobRV (-)RNA, in contrast, replacing sets of phosphodiester bonds with phosphorothioate diester bonds, even at the junction, did not greatly slow autolytic processing or spontaneous ligation, the usual reactions of the unmodified RNA. In the ligation reaction STobRV (-)RNA directed the formation of an ApG phosphorothioate diester bond.     

Synergy between transcription and mRNA processing events

Processing of eukaryotic pre-mRNAs is an important step for the translation of proteins. These processing events include the addition of a cap structure at the 5' terminus of the pre-mRNA, the splicing out of introns and the acquisition of a polyadenosine tail at the 3' terminus of the pre-mRNA. It has now become apparent that the RNA processing events can significantly influence each other. RNA polymerase II appears as a key player in these processes, cooperating with numerous processing factors that are involved in capping, splicing, and polyadenylation. More specifically, the carboxyterminal domain of the large subunit of the enzyme plays a critical role in coordination of the processing events. The number of interactions between the various RNA processing events identified so far reflects the complexity of these reactions. As more studies focus on these interactions, additional links and cellular partners will undoubtedly be discovered.     

Complete replication of poliovirus in vitro: preinitiation RNA replication complexes require soluble cellular factors for the synthesis of VPg-linked RNA.

Translation of poliovirion RNA in HeLa S10 extracts resulted in the formation of RNA replication complexes which catalyzed the asymmetric replication of poliovirus RNA. Synthesis of poliovirus RNA was detected in unfractionated HeLa S10 translation reactions and in RNA replication complexes isolated from HeLa S10 translation reactions by pulse-labeling with [32P]CTP. The RNA replication complexes formed in vitro contained replicative-intermediate RNA and were enriched in viral protein 3CD and the membrane-associated viral proteins 2C, 2BC, and 3AB. Genome-length poliovirus RNA covalently linked to VPg was synthesized in large amounts by the replication complexes. RNA replication was highly asymmetric, with predominantly positive-polarity RNA products. Both anti-VPg antibody and guanidine HCl inhibited RNA replication and virus formation in the HeLa S10 translation reactions without affecting viral protein synthesis. The inhibition of RNA synthesis by guanidine was reversible. The reversible nature of guanidine inhibition was used to demonstrate the formation of preinitiation RNA replication complexes in reaction mixes containing 2 mM guanidine HCl. Preinitiation complexes sedimented upon centrifugation at 15,000 x g and initiated RNA replication upon their resuspension in reaction mixes lacking guanidine. Initiation of RNA synthesis by preinitiation complexes did not require active protein synthesis or the addition of soluble viral proteins. Initiation of RNA synthesis by preinitiation complexes, however, was absolutely dependent on soluble HeLa cytoplasmic factors. Preinitiation complexes also catalyzed the formation of infectious virus in reaction mixes containing exogenously added capsid proteins. The titer of infectious virus produced in such trans-encapsidation reactions reached 4 x 10(7) PFU/ml. The HeLa S10 translation-RNA replication reactions represent an efficient in vitro system for authentic poliovirus replication, including protein synthesis, polyprotein processing, RNA replication, and virus assembly.     

RNA recognition by 3'-to-5' exonucleases: the substrate perspective

The 3'-to-5' exonucleolytic decay and processing of a variety of RNAs is an essential feature of RNA metabolism in all cells. The 3'-5' exonucleases, and in particular the exosome, are involved in a large number of pathways from 3' processing of rRNA, snRNA and snoRNA, to decay of mRNAs and mRNA surveillance. The potent enzymes performing these reactions are regulated to prevent processing of inappropriate substrates whilst mature RNA molecules exhibit several attributes that enable them to evade 3'-5' attack. How does an enzyme perform such selective activities on different substrates? The goal of this review is to provide an overview and perspective of available data on the underlying principles for the recognition of RNA substrates by 3'-to-5' exonucleases.     

Structure and function of the hairpin ribozyme

The hairpin ribozyme belongs to the family of small catalytic RNAs that cleave RNA substrates in a reversible reaction that generates 2',3'-cyclic phosphate and 5'-hydroxyl termini. The hairpin catalytic motif was discovered in the negative strand of the tobacco ringspot virus satellite RNA, where hairpin ribozyme-mediated self-cleavage and ligation reactions participate in processing RNA replication intermediates. The self-cleaving hairpin, hammerhead, hepatitis delta and Neurospora VS RNAs each adopt unique structures and exploit distinct kinetic and catalytic mechanisms despite catalyzing the same chemical reactions. Mechanistic studies of hairpin ribozyme reactions provided early evidence that, like protein enzymes, RNA enzymes are able to exploit a variety of catalytic strategies. In contrast to the hammerhead and Tetrahymena ribozyme reactions, hairpin-mediated cleavage and ligation proceed through a catalytic mechanism that does not require direct coordination of metal cations to phosphate or water oxygens. The hairpin ribozyme is a better ligase than it is a nuclease while the hammerhead reaction favors cleavage over ligation of bound products by nearly 200-fold. Recent structure-function studies have begun to yield insights into the molecular bases of these unique features of the hairpin ribozyme.     

Coupled translation and replication of poliovirus RNA in vitro: synthesis of functional 3D polymerase and infectious virus.

Poliovirus RNA polymerase and infectious virus particles were synthesized by translation of virion RNA in vitro in HeLa S10 extracts. The in vitro translation reactions were optimized for the synthesis of the viral proteins found in infected cells and in particular the synthesis of the viral polymerase 3Dpol. There was a linear increase in the amount of labeled protein synthesized during the first 6 h of the reaction. The appearance of 3Dpol in the translation products was delayed because of the additional time required for the proteolytic processing of precursor proteins. 3Dpol was first observed at 1 h in polyacrylamide gels, with significant amounts being detected at 6 h and later. Initial attempts to assay for polymerase activity directly in the translation reaction were not successful. Polymerase activity, however, was easily detected by adding a small amount (3 microliters) of translation products to a standard polymerase assay containing poliovirion RNA. Full-length minus-strand RNA was synthesized in the presence of an oligo(U) primer. In the absence of oligo(U), product RNA about twice the size of virion RNA was synthesized in these reactions. RNA stability studies and plaque assays indicated that a significant fraction of the input virion RNA in the translation reactions was very stable and remained intact for 20 h or more. Plaque assays indicated that infectious virus was synthesized in the in vitro translation reactions. Under optimal conditions, the titer of infectious virus produced in the in vitro translation reactions was greater than 100,000 PFU/ml. Virus was first detected at 6 h and increased to maximum levels by 12 h. Overall, the kinetics of poliovirus replication (protein synthesis, polymerase activity, and virus production) observed in the HeLa S10-initiation factor in vitro translation reactions were similar to those observed in infected cells.     

Genetic interactions suggest multiple distinct roles of the arch and core helicase domains of Mtr4 in Rrp6 and exosome function

The RNA exosome is responsible for a wide variety of RNA processing and degradation reactions. The activity and specificity of the RNA exosome is thought to be controlled by a number of cofactors. Mtr4 is an essential RNA-dependent adenosine triphosphatase that is required for all of the nuclear functions of the RNA exosome. The crystal structure of Mtr4 uncovered a domain that is conserved in the RNA exosome cofactors Mtr4 and Ski2 but not in other helicases, suggesting it has an important role related to exosome activation. Rrp6 provides the nuclear exosome with one of its three nuclease activities, and previous findings suggested that the arch domain is specifically required for Rrp6 functions. Here, we report that the genetic interactions between the arch domain of Mtr4 and Rrp6 cannot be explained by the arch domain solely acting in Rrp6-dependent processing reactions. Specifically, we show that the arch domain is not required for all Rrp6 functions, and that the arch domain also functions independently of Rrp6. Finally, we show that the arch domain of Ski2, the cytoplasmic counterpart of Mtr4, is required for Ski2’s function, thereby confirming that the arch domains of these cofactors function independently of Rrp6.     

Catalysis by RNA

Until the discovery of catalytic RNA, the notion that all enzymes are proteins had seemed incontrovertible. Now the existence of RNA enzymes has been confirmed in a variety of contexts. What is known about the chemistry of RNA-catalyzed reactions is reviewed below, with particular attention to the self-splicing rRNA intron of Tetrahymena thermophila and the processing of pre-tRNA molecules by RNase P.