Hierarchy of polyadenylation site usage by bovine papillomavirus in transformed mouse cells.

The great majority of viral mRNAs in mouse C127 cells transformed by bovine papillomavirus type 1 (BPV) have a common 3' end at the early polyadenylation site which is 23 nucleotides (nt) downstream of a canonical poly(A) consensus signal. Twenty percent of BPV mRNA from productively infected cells bypasses the early polyadenylation site and uses the late polyadenylation site approximately 3,000 nt downstream. To inactivate the BPV early polyadenylation site, the early poly(A) consensus signal was mutated from AAUAAA to UGUAAA. Surprisingly, this mutation did not result in significant read-through expression of downstream RNA. Rather, RNA mapping and cDNA cloning experiments demonstrate that virtually all of the mutant RNA is cleaved and polyadenylated at heterogeneous sites approximately 100 nt upstream of the wild-type early polyadenylation site. In addition, cells transformed by wild-type BPV harbor a small population of mRNAs with 3' ends located in this upstream region. These experiments demonstrate that inactivation of the major poly(A) signal induces preferential use of otherwise very minor upstream poly(A) sites. Mutational analysis suggests that polyadenylation at the minor sites is controlled, at least in part, by UAUAUA, an unusual variant of the poly(A) consensus signal approximately 25 nt upstream of the minor polyadenylation sites. These experiments indicate that inactivation of the major early polyadenylation signal is not sufficient to induce expression of the BPV late genes in transformed mouse cells.     

Modulation of formation of the 3’-end of the human argininosuccinate synthetase mRNA by GT-repeat polymorphism

Microsatellites are abundant in the human genome and may acquire context-dependent functions. A highly polymorphic GT microsatellite is located downstream of the poly(A) signal of the human argininosuccinate synthetase (ASS1) gene. The ASS1 participates in urea and nitric oxide production and is a rate-limiting enzyme in arginine biosynthesis. To examine possible involvement of the GT microsatellite in ASS1 mRNA 3’-end formation, ASS1 minigene constructs were used in transient transfection for assessment of poly(A) site usage by S1 nuclease mapping. Synthesis of the major human ASS1 mRNA is found to be controlled by two consecutive non-canonical poly(A) signals, UAUAAA and AUUAAA, located 7 nucleotides apart where a U-rich sequence and the GU microsatellite serve as their respective downstream GU/U-rich elements. Moreover, AUUAAA utilization is affected by the GU-repeat number possibly leading to differential regulation of ASS1 polyadenylation in individuals with different repeat numbers. Interestingly, the less efficient UAUAAA motif is noted to be the major ASS1 poly(A) signal possibly as a result of an indispensable downstream U-rich element and restricted utilization of the AUUAAA motif by the presence of extended GU-repeats. The UAUAAA motif and the GT microsatellite are conserved only in primates whereas AUUAAA motif is present in all mammals analyzed. The suboptimal UAUAAA motif and the utilization of the polymorphic GT microsatellite as polyadenylation signal of the ASS1 gene may be used as a strategy in primates to modulate ASS1 level in response to interactions of genetic and environmental factors.     

Molecular cloning and nucleotide sequence of cDNA coding for rat brain cholecystokinin precursor

A mixture of 14-mer oligodeoxynucleotides was used for the screening of a cDNA clone coding for a cholecystokinin (CCK) precursor from a cDNA library for rat brain microsomal poly(A)RNA. The longest insert is 718 bp long which was verified to contain a nearly full-length cDNA sequence coding for rat CCK precursor, because the size of CCK mRNA was estimated to be about 850 bases long by Northern blotting analysis. Sequence analysis revealed 110 bp in the 5'-untranslated region, 345 bp in the amino acid coding region corresponding to the CCK precursor and 263 bp in the 3'-noncoding region which contains polyadenylation signal AUUAAA and the poly(A) sequence. The precursor may contain a 28 amino acid signal peptide and 12 additional amino acids at the carboxyl terminus.     

Polyadenylation of mRNA: minimal substrates and a requirement for the 2'' hydroxyl of the U in AAUAAA.

mRNA-specific polyadenylation can be assayed in vitro by using synthetic RNAs that end at or near the natural cleavage site. This reaction requires the highly conserved sequence AAUAAA. At least two distinct nuclear components, an AAUAAA specificity factor and poly(A) polymerase, are required to catalyze the reaction. In this study, we identified structural features of the RNA substrate that are critical for mRNA-specific polyadenylation. We found that a substrate that contained only 11 nucleotides, of which the first six were AAUAAA, underwent AAUAAA-specific polyadenylation. This is the shortest substrate we have used that supports polyadenylation: removal of a single nucleotide from either end of this RNA abolished the reaction. Although AAUAAA appeared to be the only strict sequence requirement for polyadenylation, the number of nucleotides between AAUAAA and the 3' end was critical. Substrates with seven or fewer nucleotides beyond AAUAAA received poly(A) with decreased efficiency yet still bound efficiently to specificity factor. We infer that on these shortened substrates, poly(A) polymerase cannot simultaneously contact the specificity factor bound to AAUAAA and the 3' end of the RNA. By incorporating 2'-deoxyuridine into the U of AAUAAA, we demonstrated that the 2' hydroxyl of the U in AAUAAA was required for the binding of specificity factor to the substrate and hence for poly(A) addition. This finding may indicate that at least one of the factors involved in the interaction with AAUAAA is a protein.     

Conditional defect in mRNA 3'' end processing caused by a mutation in the gene for poly(A) polymerase.

Maturation of most eukaryotic mRNA 3' ends requires endonucleolytic cleavage and polyadenylation of precursor mRNAs. To further understand the mechanism and function of mRNA 3' end processing, we identified a temperature-sensitive mutant of Saccharomyces cerevisiae defective for polyadenylation. Genetic analysis showed that the polyadenylation defect and the temperature sensitivity for growth result from a single mutation. Biochemical analysis of extracts from this mutant shows that the polyadenylation defect occurs at a step following normal site-specific cleavage of a pre-mRNA at its polyadenylation site. Molecular cloning and characterization of the wild-type allele of the mutated gene revealed that it (PAP1) encodes a previously characterized poly(A) polymerase with unknown RNA substrate specificity. Analysis of mRNA levels and structure in vivo indicate that shift of growing, mutant cells to the nonpermissive temperature results in the production of poly(A)-deficient mRNAs which appear to end at their normal cleavage sites. Interestingly, measurement of the rate of protein synthesis after the temperature shift shows that translation continues long after the apparent loss of polyadenylated mRNA. Our characterization of the pap1-1 defect implicates this gene as essential for mRNA 3' end formation in S. cerevisiae.     

Sedimentation analysis of polyadenylation-specific complexes.

Precursor RNA containing the adenovirus L3 polyadenylation site is assembled into a 50S complex upon incubation with HeLa nuclear extract at 30 degrees C. The cofactor and sequence requirements for 50S complex formation are similar to those of the in vitro polyadenylation reaction. Assembly of this complex requires ATP but is not dependent upon synthesis of a poly(A) tract. In addition, a 50S complex does not form on substrate RNA in which the AAUAAA hexanucleotide upstream of the poly(A) site has been mutated to AAGAAA or on RNA in which sequences between +5 and +48 nucleotides downstream of the site have been removed. These mutations also prevent in vitro processing of substrate RNA. Kinetic studies suggest that the 50S complex is an intermediate in the polyadenylation reaction. It forms at an early stage in the reaction and at later times contains both poly(A)+ RNA as well as unreacted precursor. U-type small nuclear ribonucleoprotein particles are components of the 50S complex, as shown by immunoprecipitation with antiserum specific to the trimethyl cap of these small nuclear RNAs.     

Cloning, Sequencing, Expression, and Purification of the C Isozyme of Mouse Phosphofructokinase

The cDNA of mouse phosphofructo-1-kinase isozyme C was cloned and sequenced. The coding region translates into a protein of 85,473 Da containing 785 amino acids. The cDNA includes 57 base pairs of a 5'-untranslated region and a 3' untranslated region of 284 base pairs containing a polyadenylation signal, AUUAAA, located 17 bases upstream from the poly(A) tail. The cDNA was ligated into a pET vector and transformed into a pfk(-) strain of Escherichia coli (DF1020) that contained the pLysS plasmid and an integrated lambda DE3 prophage that includes a single copy of the gene for T7 RNA polymerase under control of the inducible LacUV5 promoter. Conditions for maximum induction of soluble enzyme activity was developed to produce up to 2400 units of soluble enzyme activity per liter of growth medium. The enzyme could be purified to homogeneity with a yield of approximately 60% by a single purification step on ATP-Sepharose.     

Characterization of specific protein-RNA complexes associated with the coupling of polyadenylation and last-intron removal

Polyadenylation and splicing are highly coordinated on substrate RNAs capable of coupled polyadenylation and splicing. Individual elements of both splicing and polyadenylation signals are required for the in vitro coupling of the processing reactions. In order to understand more about the coupling mechanism, we examined specific protein-RNA complexes formed on RNA substrates, which undergo coupled splicing and polyadenylation. We hypothesized that formation of a coupling complex would be adversely affected by mutations of either splicing or polyadenylation elements known to be required for coupling. We defined three specific complexes (A(C)', A(C), and B(C)) that form rapidly on a coupled splicing and polyadenylation substrate, well before the appearance of spliced and/or polyadenylated products. The A(C)' complex is formed by 30 s after mixing, the A(C) complex is formed between 1 and 2 min after mixing, and the B(C) complex is formed by 2 to 3 min after mixing. A(C)' is a precursor of A(C), and the A(C)' and/or A(C) complex is a precursor of B(C). Of the three complexes, B(C) appears to be a true coupling complex in that its formation was consistently diminished by mutations or experimental conditions known to disrupt coupling. The characteristics of the A(C)' complex suggest that it is analogous to the spliceosomal A complex, which forms on splicing-only substrates. Formation of the A(C)' complex is dependent on the polypyrimidine tract. The transition from A(C)' to A(C) appears to require an intact 3'-splice site. Formation of the B(C) complex requires both splicing elements and the polyadenylation signal. A unique polyadenylation-specific complex formed rapidly on substrates containing only the polyadenylation signal. This complex, like the A(C)' complex, formed very transiently on the coupled splicing and polyadenylation substrate; we suggest that these two complexes coordinate, resulting in the B(C) complex. We also suggest a model in which the coupling mechanism may act as a dominant checkpoint in which aberrant definition of one exon overrides the normal processing at surrounding wild-type sites.     

细菌RNA聚腺苷酸化修饰及其调控机制与生理作用

聚腺苷酸化 (polyadenylation) 是指在底物RNA的3′-端加上一段聚腺苷酸残基的转录后修饰作用。1971年,第一次发现真核生物mRNA的3′-端存在多聚腺苷酸 (poly (A)) 尾,它保护mRNA免受核酸外切酶攻击,且对于转录终止、mRNA运输及翻译都起到重要作用,学者们一度将该现象认为是真核细胞mRNA的特征之一。时至今日,细菌RNA聚腺苷酸化现象的发现引起了学术界的高度重视,大量的研究结果不仅证明了该种修饰在细菌中普遍存在,而且发现其在细菌RNA的加工、降解及质量监控中扮演重要的角色;然而,与真核生物不同的是,在原核生物中该修饰倾向于使RNA去稳定化,即加速RNA的降解。本文综述了近年来细菌中RNA聚腺苷酸化修饰及其调控机制与生理作用的研究进展。     

Contributions of Trf4p- and Trf5p-dependent polyadenylation to the processing and degradative functions of the yeast nuclear exosome

The nuclear exosome is involved in a large number of RNA processing and surveillance pathways. RNase III cleavage intermediates destined to be 3'-processed or degraded can be detected when the Rrp6p subunit of the nuclear exosome is absent. Here we show that these processing and degradation intermediates are polyadenylated, and that their polyadenylation is dependent on the activity of Trf4p and Trf5p, two variant poly(A) polymerases. Polyadenylation of cleavage intermediates was inhibited when Trf4p was absent, and reduced to various extents in the absence of Trf5p, suggesting that these two poly(A) polymerases play functionally distinct roles in the polyadenylation of these RNA species. Finally, in the absence of Trf4p, we observed 3'-extended forms of the U4 snRNA that are similar to those observed in the absence of Rrp6p. These results suggest that polyadenylation of RNA processing intermediates plays a functional role in RNA processing pathways and is not limited to RNA surveillance functions.     

Identification of a complex associated with processing and polyadenylation in vitro of herpes simplex virus type 1 thymidine kinase precursor RNA.

Cleavage and polyadenylation of substrate RNAs containing the herpes simplex virus type 1 (HSV-1) thymidine kinase (tk) gene polyadenylation signal region were examined in HeLa cell nuclear extract. 3'-End RNA processing was accurate and efficient and required ATP and Mg2+. Cleavage, but not polyadenylation, occurred in the presence of EDTA or when ATP was replaced with 3' dATP (cordycepin) or AMP(CH2)PP, a nonhydrolyzable analog of ATP. Processing in vitro and in vivo showed the same signal element requirements: a series of substrates containing linker scanning, internal deletion, and small insertion mutations was processed with the same relative efficiencies and at the same sites in vitro and in vivo. A complex involved in 3'-end RNA processing was identified by gel mobility shift analysis. This complex formed rapidly, reached a maximum level after 20 to 30 min, and was much reduced after 2 h. Very little complex was formed at 0 degree C or with substrates lacking a polyadenylation signal. Entry of 32P-labeled tk substrate into the complex could be prevented by addition of excess 35S-labeled tk or adenovirus L3 precursor RNAs. Competition was not observed with tk RNAs lacking a complete polyadenylation signal.     

A multicomponent complex is required for the AAUAAA-dependent cross-linking of a 64-kilodalton protein to polyadenylation substrates.

A 64-kilodalton (kDa) polypeptide that is cross-linked by UV light specifically to polyadenylation substrate RNAs containing a functional AAUAAA element has been identified previously. Fractionated HeLa nuclear components that can be combined to regenerate efficient and accurate polyadenylation in vitro have now been screened for the presence of the 64-kDa protein. None of the individual components contained an activity which could generate the 64-kDa species upon UV cross-linking in the presence of substrate RNA. It was necessary to mix two components, cleavage stimulation factor and specificity factor, to reconstitute 64-kDa protein-RNA cross-linking. The addition of cleavage factors to this mixture very efficiently reconstituted the AAUAAA-specific 64-kDa protein-RNA interaction. The 64-kDa protein, therefore, is present in highly purified, reconstituted polyadenylation reactions. However, it is necessary to form a multicomponent complex to efficiently cross-link the protein to a substrate RNA.     

In vitro cleavage of the simian virus 40 early polyadenylation site adjacent to a required downstream TG sequence.

Exogenous RNA containing the simian virus 40 early polyadenylation site was efficiently and accurately polyadenylated in in vitro nuclear extracts. Correct cleavage required ATP. In the absence of ATP, nonpoly(A)+ products accumulated which were 18 to 20 nucleotides longer than the RNA generated by correct cleavage; the longer RNA terminated adjacent to the downstream TG element required for polyadenylation. In the presence of ATP analogs, alternate cleavage was not observed; instead, correct cleavage without poly(A) addition occurred. ATP-independent cleavage of simian virus 40 early RNA had many of the same properties as correct cleavage including requirements for an intact AAUAAA element, a proximal 3' terminus, and extract small nuclear ribonucleoproteins. This similarity in reaction parameters suggested that ATP-independent cleavage is an activity of the normal polyadenylation machinery. The ATP-independent cleavage product, however, did not behave as an intermediate in polyadenylation. The alternate RNA did not preferentially chase into correctly cleaved material upon readdition of ATP; instead, poly(A) was added to the 3' terminus of the cleaved RNA during a chase. Purified ATP-independent cleavage RNA, however, was a substrate for correct cleavage when reintroduced into the nuclear extract. Thus, alternate cleavage of polyadenylation sites adjacent to a required downstream sequence element is directed by the polyadenylation machinery in the absence of ATP.     

Processing at immunoglobulin polyadenylation sites in lymphoid cell extracts.

We have developed an in vitro system for polyadenylation of RNA substrates in cell-free nuclear extracts prepared from murine cells of lymphoid origin. RNA substrates containing the adenovirus L3, murine immunoglobulin (IgM) secreted and membrane polyadenylation sites were accurately polyadenylated in these extracts. Kinetic analysis showed that the rate of polyadenylation in vitro responds proportionally to the substrate concentration. Quantitation of the initial rate of polyadenylation at the three sites permitted comparison of the activities of extracts prepared from HeLa cells, B cells (Wehi 231) and plasmacytoma cells (P9.37.11). From this analysis, we concluded that in all three extracts the polyadenylation activity at the L3 site was higher than that of either of the IgM sites. In contrast to the preferential utilization of the secreted site in vivo in plasmacytomas, this site was not selectively processed in plasmacytoma as compared to B cell extracts. The efficiency of polyadenylation at both IgM sites in the plasmacytoma extract was significantly lower than that in the B cell extract. The common low activity at the IgM sites in the plasmacytoma cell extract suggests that the rate-limiting step for polyadenylation at these two sites differs from that at the L3 site.