Further analysis of cytoplasmic polyadenylation in Xenopus embryos and identification of embryonic cytoplasmic polyadenylation element-binding proteins.

Early development in Xenopus laevis is programmed in part by maternally inherited mRNAs that are synthesized and stored in the growing oocyte. During oocyte maturation, several of these messages are translationally activated by poly(A) elongation, which in turn is regulated by two cis elements in the 3' untranslated region, the hexanucleotide AAUAAA and a cytoplasmic polyadenylation element (CPE) consisting of UUUUUAU or similar sequence. In the early embryo, a different set of maternal mRNAs is translationally activated. We have shown previously that one of these, C12, requires a CPE consisting of at least 12 uridine residues, in addition to the hexanucleotide, for its cytoplasmic polyadenylation and subsequent translation (R. Simon, J.-P. Tassan, and J.D. Richter, Genes Dev. 6:2580-2591, 1992). To assess whether this embryonic CPE functions in other maternal mRNAs, we have chosen Cl1 RNA, which is known to be polyadenylated during early embryogenesis (J. Paris, B. Osborne, A. Couturier, R. LeGuellec, and M. Philippe, Gene 72:169-176, 1988). Wild-type as well as mutated versions of Cl1 RNA were injected into fertilized eggs and were analyzed for cytoplasmic polyadenylation at times up to the gastrula stage. This RNA also required a poly(U) CPE for cytoplasmic polyadenylation in embryos, but in this case the CPE consisted of 18 uridine residues. In addition, the timing and extent of cytoplasmic poly(A) elongation during early embryogenesis were dependent upon the distance between the CPE and the hexanucleotide. Further, as was the case with Cl2 RNA, Cl1 RNA contains a large masking element that prevents premature cytoplasmic polyadenylation during oocyte maturation. To examine the factors that may be involved in the cytoplasmic polyadenylation of both C12 and C11 RNAs, we performed UV cross-linking experiments in egg extracts. Two proteins with sizes of ~36 and ~45 kDa interacted specifically with the CPEs of both RNAs, although they bound preferentially to the C12 CPE. The role that these proteins might play in cytoplasmic polyadenylation is discussed.     

Multiple sequence elements and a maternal mRNA product control cdk2 RNA polyadenylation and translation during early Xenopus development.

Cytoplasmic poly(A) elongation is one mechanism that regulates translational recruitment of maternal mRNA in early development. In Xenopus laevis, poly(A) elongation is controlled by two cis elements in the 3' untranslated regions of responsive mRNAs: the hexanucleotide AAUAAA and a U-rich structure with the general sequence UUUUUAAU, which is referred to as the cytoplasmic polyadenylation element (CPE). B4 RNA, which contains these sequences, is polyadenylated during oocyte maturation and maintains a poly(A) tail in early embryos. However, cdk2 RNA, which also contains these sequences, is polyadenylated during maturation but deadenylated after fertilization. This suggests that cis-acting elements in cdk2 RNA signal the removal of the poly(A) tail at this time. By using poly(A) RNA-injected eggs, we showed that two elements which reside 5' of the CPE and 3' of the hexanucleotide act synergistically to promote embryonic deadenylation of this RNA. When an identical RNA lacking a poly(A) tail was injected, these sequences also prevented poly(A) addition. When fused to CAT RNA, the cdk2 3' untranslated region, which contains these elements, as well as the CPE and the hexanucleotide, promoted poly(A) addition and enhanced chloramphenicol acetyltransferase activity during maturation, as well as repression of these events after fertilization. Incubation of fertilized eggs with cycloheximide prevented the embryonic inhibition of cdk2 RNA polyadenylation but did not affect the robust polyadenylation of B4 RNA. This suggests that a maternal mRNA, whose translation occurs only after fertilization, is necessary for the cdk2 deadenylation or inhibition of RNA polyadenylation. This was further suggested when poly(A)+ RNA isolated from two-cell embryos was injected into oocytes that were then allowed to mature. Such oocytes became deficient for cdk2 RNA polyadenylation but remained proficient for B4 RNA polyadenylation. These data show that CPE function is developmentally regulated by multiple sequences and factors.     

RNA-RNA recombination in Sindbis virus: roles of the 3' conserved motif, poly(A) tail, and nonviral sequences of template RNAs in polymerase recognition and template switching.

Sindbis virus (SIN), a mosquito-transmitted animal RNA virus, carries a 11.7-kb positive-sense RNA genome which is capped and polyadenylated. We recently reported that the SIN RNA-dependent RNA polymerase (RdRp) could initiate negative-strand RNA synthesis from a 0.3-kb 3'-coterminal SIN RNA fragment and undergo template switching in vivo (M. Hajjou, K. R. Hill, S. V. Subramaniam, J. Y. Hu, and R. Raju, J. Virol. 70:5153-5164, 1996). To identify and characterize the viral and nonviral sequences which regulate SIN RNA synthesis and recombination, a series of SIN RNAs carrying altered 3' ends were tested for the ability to produce infectious virus or to support recombination in BHK cells. The major findings of this report are as follows: (i) the 3'-terminal 20-nucleotides (nt) sequence along with the abutting poly(A) tail of the SIN genome fully supports negative-strand synthesis, genome replication, and template switching; (ii) a full-length SIN RNA carrying the 3'-terminal 24 nt but lacking the poly(A) tail is noninfectious; (iii) SIN RNAs which carry 3' 64 nt or more without the poly(A) tail are infectious and regain their poly(A) tail in vivo; (iv) donor templates lacking the poly(A) tail do not support template switching; (v) full-length SIN RNAs lacking the poly(A) tail but carrying 3' nonviral extensions, although debilitated to begin with, evolve into rapidly growing poly(A)-carrying mutants; (vi) poly(A) or poly(U) motifs positioned internally within the acceptor templates, in the absence of other promoter elements within the vicinity, do not induce the jumping polymerase to reinitiate at these sites; and (vii) the junction site selection on donor templates occurs independently of the sequences around the acceptor sites. In addition to furthering our understanding of RNA recombination, these studies give interesting clues as to how the alphavirus polymerase interacts with its 3' promoter elements of genomic RNA and nonreplicative RNAs. This is the first report that an in vitro-synthesized alphavirus RNA lacking a poly(A) tail can initiate infection and produce 3' polyadenylated viral genome in vivo.     

Accurate cleavage and polyadenylation of exogenous RNA substrate

Purified precursor RNA containing the L3 polyadenylation site of late adenovirus 2 mRNA is accurately cleaved and polyadenylated when incubated with nuclear extract from HeLa cells. The reaction is very efficient; 75% of the precursor is correctly processed. Cleavage is rapidly followed by polymerization of an initial poly(A) tract of approximately 130 nucleotides. Additional adenosine residues are added during further incubation. In the presence of the ATP analog alpha-beta-methylene-adenosine 5' triphosphate, the precursor RNA is cleaved but not polyadenylated, suggesting that processing is not coupled to the synthesis of the initial poly(A) tract. In the absence of free Mg2+, a small RNA of approximately 46 nucleotides is stabilized against degradation. Fingerprint analysis suggests this RNA is produced by endonucleolytic cleavage at the L3 site. Like the in vitro splicing reaction, the in vitro polyadenylation reaction is inhibited by adding antiserum against the small nuclear ribonucleoprotein particle containing U1 RNA.     

Polyadenylation regulates the stability of Trypanosoma brucei mitochondrial RNAs

Polyadenylation of RNAs plays a critical role in modulating rates of RNA turnover and ultimately in controlling gene expression in all systems examined to date. In mitochondria, the precise mechanisms by which RNAs are degraded, including the role of polyadenylation, are not well understood. Our previous in organello pulse-chase experiments suggest that poly(A) tails stimulate degradation of mRNAs in the mitochondria of the protozoan parasite Trypanosoma brucei (Militello, K. T., and Read, L. K. (2000) Mol. Cell. Biol. 21, 731-742). In this report, we developed an in vitro assay to directly examine the effects of specific 3'-sequences on RNA degradation. We found that a salt-extracted mitochondrial membrane fraction preferentially degraded polyadenylated mitochondrially and non-mitochondrially encoded RNAs over their non-adenylated counterparts. A poly(A) tail as short as 5 nucleotides was sufficient to stimulate rapid degradation, although an in vivo tail length of 20 adenosines supported the most rapid decay. A poly(U) extension did not promote rapid RNA degradation, and RNA turnover was slowed by the addition of uridine residues to the poly(A) tail. To stimulate degradation, the poly(A) element must be located at the 3' terminus of the RNA. Finally, we demonstrate that degradation of polyadenylated RNAs occurs in the 3' to 5' direction through the action of a hydrolytic exonuclease. These experiments demonstrate that the poly(A) tail can act as a cis-acting element to facilitate degradation of T. brucei mitochondrial mRNAs.     

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。     

An RNA secondary structure juxtaposes two remote genetic signals for human T-cell leukemia virus type I RNA 3'-end processing.

Sequence analysis of the human T-cell leukemia virus type I (HTLV-I) long terminal repeat (LTR) does not reveal a polyadenylation consensus sequence, AAUAAA, close to the polyadenylation site at the 3' end of the viral RNA. Using site-directed mutagenesis, we demonstrated that two cis-acting signals are required for efficient RNA processing in HTLV-I LTR: (i) a remote AAUAAA hexamer at a distance of 276 nucleotides upstream of the polyadenylation site, and (ii) the 20-nucleotide GU-rich sequence immediately downstream from the poly(A) site. It has been postulated that the folding of RNA into a secondary structure juxtaposes the AAUAAA sequence, in a noncontiguous manner, to within 14 nucleotides of the polyadenylation site. To test this hypothesis, we introduced deletions and point mutations within the U3 and R regions of the LTR. RNA 3'-end processing occurred efficiently at the authentic HTLV-I poly(A) site after deletion of the sequences predicted to form the secondary structure. Thus, the genetic analysis supports the hypothesis that folding of the HTLV-I RNA in the U3 and R regions juxtaposes the AAUAAA sequence and the poly(A) site to the correct functional distance. This unique arrangement of RNA-processing signals is also found in the related retroviruses HTLV-II and bovine leukemia virus.