Assaying the polyadenylation state of mRNAs

The poly(A) tail present at the 3' end of most eukaryotic mRNAs can play a critical role in message translation and stability. Therefore, identifying alterations in poly(A) tail length can yield important insights into an mRNA's function and subsequent physiological impact. Here, we present three methods for assaying polyadenylation of a specific mRNA in the context of total cellular RNA. The first method described, oligo(dT)/RNase H-Northern analysis, is the classic labor-intensive assay for polyadenylation and is included for historical reference and as a potential experimental control for the poly(A) test (PAT) assays described subsequently. The PAT methods-rapid amplification of cDNA ends-PAT (RACE-PAT), and ligase-mediated PAT (LM-PAT)-are polymerase chain reaction-driven assays that allow speed, sensitivity, and length quantitation. The PAT assays can be conducted in a single day and can readily detect the poly(A) status of an mRNA present in subnanogram quantities of total cellular RNA.     

Regulated polyadenylation of clam maternal mRNAs in vitro

During meiotic maturation of Spisula oocytes, maternal mRNAs undergo changes in translation and in the length of their poly(A) tails. In general, those mRNAs that are translationally activated, i.e., unmasked become polyadenylated, while deactivated mRNAs lose their poly(A) tails. The activated class of mRNAs encode ribonucleotide reductase, cyclins A and B and histone H3, while the proteins that stop being made include tubulin and actin. Previously, we demonstrated that mRNA-specific unmasking can be brought about in vitro by preventing the interaction of protein(s) with central portions of the 3′ noncoding regions (masking regions) of ribonucle-otide reductase and cyclin A mRNAs. In this report, we show that clam egg extracts are capable of sequence-specific polyadenylation of added RNAs since the 3′ untranslated regions (UTRs) of ribonu-cleotide reductase and histone H3 mRNAs are polyadenylated, while that of actin mRNA is not. In contrast, oocyte extracts, as in vivo, are essentially devoid of polyadenylation activity. We present an initial characterisation of the cis-acting sequences in the 3′ UTR of ribonucleotide reductase mRNA required for polyadenylation. The results suggest that the sequences for cytoplasmic polyadenylation are more complex and extensive than those determined in vertebrates and that they may partly overlap with the masking regions. © 1993 Wiley-Liss, Inc.     

A novel method for constructing murine cDNA library enriched with maternal mRNAs exhibiting de novo independent post-fertilization polyadenylation

Recently, mouse maternal mRNAs such as SSEC-D, Spin, beta-catenin, Ptp4a1, and Maid have been found to exhibit de novo independent polyadenylation after fertilization. To obtain an overall picture of post-fertilization polyadenylation events, we developed a novel method for constructing murine fertilized egg cDNA library enriched with cDNAs exhibiting de novo independent polyadenylation. As a pilot study, we isolated at least four new maternal mRNAs exhibiting extension of poly(A) tail in fertilized 1-cell eggs. Moreover, various types of polyadenylation of maternal RNAs were observed at this stage, suggesting the presence of novel mechanisms for regulating the length of poly(A) tails of maternal mRNA. This is the first report of successful construction of a cDNA library enriched with newly polyadenylated maternal mRNAs derived from post-fertilized mouse eggs. This cDNA library will be useful for molecular analysis of the mechanisms underlying post-fertilization polyadenylation of mammalian maternal RNAs.     

A small nuclear ribonucleoprotein associates with the AAUAAA polyadenylation signal in vitro

RNAs containing the polyadenylation sites for adenovirus L3 or E2a mRNA or for SV40 early or late mRNA are substrates for cleavage and poly(A) addition in an extract of HeLa cell nuclei. When polyadenylation reactions are probed with ribonuclease T1 and antibodies directed against either the Sm protein determinant or the trimethylguanosine cap structure at the 5' end of U RNAs in small nuclear ribonucleoproteins, RNA fragments containing the AAUAAA polyadenylation signal are immunoprecipitated. The RNA cleavage step that occurs prior to poly(A) addition is inhibited by micrococcal nuclease digestion of the nuclear extract. The immunoprecipitation of fragments containing the AAUAAA sequence can be altered, but not always abolished, by pretreatment with micrococcal nuclease. We discuss the involvement of small nuclear ribonucleoproteins in the cleavage and poly(A) addition reactions that form the 3' ends of most eukaryotic mRNAs.     

Meiotic maturation in Xenopus requires polyadenylation of multiple mRNAs.

Cytoplasmic polyadenylation of specific mRNAs commonly is correlated with their translational activation during development. Here, we focus on links between cytoplasmic polyadenylation, translational activation and the control of meiotic maturation in Xenopus oocytes. We manipulate endogenous c-mos mRNA, which encodes a protein kinase that regulates meiotic maturation. We determined that translational activation of endogenous c-mos mRNA requires a long poly(A) tail per se, rather than the process of polyadenylation. For this, we injected 'prosthetic' poly(A)_synthetic poly(A) tails designed to attach by base pairing to endogenous c-mos mRNA that has had its own polyadenylation signals removed. This prosthetic poly(A) tail activates c-mos translation and restores meiotic maturation in response to progesterone. Thus the role of polyadenylation in activating c-mos mRNA differs from its role in activating certain other mRNAs, for which the act of polyadenylation is required. In the absence of progesterone, prosthetic poly(A) does not stimulate c-mos expression, implying that progesterone acts at additional steps to elevate c-Mos protein. By using a general inhibitor of polyadenylation together with prosthetic poly(A), we demonstrate that these additional steps include polyadenylation of at least one other mRNA, in addition to that of c-mos mRNA. These other mRNAs, encoding regulators of meiotic maturation, act upstream of c-Mos in the meiotic maturation pathway.     

Variability of polyadenylation sites in mRNAs from human fetal liver

cDNA libraries of human fetal liver were constructed in pBR322 and λgt10 vectors. The libraries were screened for liver-specific clones by differential hybridization. This procedure revealed 25 and 32 liver-specific clones in plasmid and phage libraries, respectively. The majority of these clones were represented with serum albumin, fetal ^Gγ-globin and ^Aγ-globin cDNA inserts. Three types of 3′-non-coding region were found in 5 sequenced albumin cDNAs. In one type mRNA the distance between the AATAAA signal and polyadenylation site was 15 nucleotides, in 2 other types this distance was 10 and 6 nucleotides. The polyadenylation site in the ^Gγ-globin cDNA was located 2 nucleotides further from AATAAA signal, while in the ^Aγ-globin cDNA it was 2 nucleotides closer to the signal as compared with the results published previously.     

A lamin-independent pathway for nuclear envelope assembly

The nuclear envelope is composed of membranes, nuclear pores, and a nuclear lamina. Using a cell-free nuclear assembly extract derived from Xenopus eggs, we have investigated how these three components interact during nuclear assembly. We find that the Xenopus embryonic lamin protein LIII cannot bind directly to chromatin or membranes when each is present alone, but is readily incorporated into nuclei when both of the components are present together in an assembly extract. We find that depleting lamin LIII from an extract does not prevent formation of an envelope consisting of membranes and nuclear pores. However, these lamin-depleted envelopes are extremely fragile and fail to grow beyond a limited extent. This suggests that lamin assembly is not required during the initial steps of nuclear envelope formation, but is required for later growth and for maintaining the structural integrity of the envelope. We also present results showing that lamins may only be incorporated into nuclei after DNA has been encapsulated within an envelope and nuclear transport has been activated. With respect to nuclear function, our results show that the presence of a nuclear lamina is required for DNA synthesis to occur within assembled nuclei.     

Regulated nuclear polyadenylation of Xenopus albumin pre-mRNA.

Cytoplasmic regulation of the length of poly(A) on mRNA is a well-characterized process involved in translational control during development. In contrast, there is no direct in vivo evidence for regulation of the length of poly(A) added during nuclear pre-mRNA processing in somatic cells. We previously reported that Xenopus serum albumin [Schoenberg et al. (1989) Mol. Endocrinol. 3, 805-815] and transferrin [Pastori et al. (1992) J. Steroid Biochem. Mol. Biol. 42, 649-657], mRNA have exceptionally short poly(A) tails ranging from 12 to 17 residues, whereas vitellogenin mRNA has long poly(A). An RT-PCR protocol was adapted to determine the length of poly(A) added onto pre-mRNA, defined here as that species bearing the terminal intron. Using this assay we show that vitellogenin pre-mRNA has the same long poly(A) tail as mature vitellogenin mRNA. In contrast, albumin pre-mRNA has the same short poly(A) as found on fully-processed albumin mRNA. These results indicate that the short poly(A) tail on albumin mRNA results from regulation of poly(A) addition during nuclear 3' processing.     

Dial tm for rescue: tmRNA engages ribosomes stalled on defective mRNAs

Ribosomes translate genetic information encoded by mRNAs into protein chains with high fidelity. Truncated mRNAs lacking a stop codon will cause the synthesis of incomplete peptide chains and stall translating ribosomes. In bacteria, a ribonucleoprotein complex composed of tmRNA, a molecule that combines the functions of tRNAs and mRNAs, and small protein B (SmpB) rescues stalled ribosomes. The SmpB-tmRNA complex binds to the stalled ribosome, allowing translation to resume at a short internal tmRNA open reading frame that encodes a protein degradation tag. The aberrant protein is released when the ribosome reaches the stop codon at the end of the tmRNA open reading frame and the fused peptide tag targets it for degradation by cellular proteases. The recently determined NMR structures of SmpB, the crystal structure of the SmpB-tmRNA complex and the cryo-EM structure of the SmpB-tmRNA-EF-Tu-ribosome complex have provided first detailed insights into the intricate mechanisms involved in ribosome rescue.     

A bipartite U1 site represses U1A expression by synergizing with PIE to inhibit nuclear polyadenylation

U1A protein negatively autoregulates itself by polyadenylation inhibition of its own pre-mRNA by binding as two molecules to a 3'UTR-located Polyadenylation Inhibitory Element (PIE). The (U1A)2-PIE complex specifically blocks U1A mRNA biosynthesis by inhibiting polyA tail addition, leading to lower mRNA levels. U1 snRNP bound to a 5'ss-like sequence, which we call a U1 site, in the 3'UTRs of certain papillomaviruses leads to inhibition of viral late gene expression via a similar mechanism. Although such U1 sites can also be artificially used to potently silence reporter and endogenous genes, no naturally occurring U1 sites have been found in eukaryotic genes. Here we identify a conserved U1 site in the human U1A gene that is, unexpectedly, within a bipartite element where the other part represses the U1 site via a base-pairing mechanism. The bipartite element inhibits U1A expression via a synergistic action with the nearby PIE. Unexpectedly, synergy is not based on stabilizing binding of the inhibitory factors to the 3'UTR, but rather is a property of the larger ternary complex. Inhibition targets the biosynthetic step of polyA tail addition rather than altering mRNA stability. This is the first example of a functional U1 site in a cellular gene and of a single gene containing two dissimilar elements that inhibit nuclear polyadenylation. Parallels with other examples where U1 snRNP inhibits expression are discussed. We expect that other cellular genes will harbor functional U1 sites.