Downstream sequence elements with different affinities for the hnRNP H/H' protein influence the processing efficiency of mammalian polyadenylation signals

Auxiliary factors likely play an important role in determining the polyadenylation efficiency of mammalian pre-mRNAs. We previously identified an auxiliary factor, hnRNP H/H′, which stimulates 3′-end processing through an interaction with sequences downstream of the core elements of the SV40 late polyadenylation signal. Using in vitro reconstitution assays we have demonstrated that hnRNP H/H′ can stimulate processing of two additional model polyadenylation signals by binding at similar relative downstream locations but with significantly different affinities. A short tract of G residues was determined to be a common property of all three hnRNP H/H′ binding sites. A survey of mammalian polyadenylation signals identified potential G-rich hnRNP H/H′ binding sites at similar downstream locations in ~34% of these signals. All of the novel G-rich elements tested were found to bind hnRNP H/H′ protein and the processing of selected signals identified in the survey was stimulated by the protein both in vivo and in vitro. Downstream G-rich tracts, therefore, are a common auxiliary element in mammalian polyadenylation signals. Sequences capable of binding hnRNP H protein with varying affinities may play a role in determining the processing efficiency of a significant number of mammalian polyadenylation signals.     

The temporal control of Wee1 mRNA translation during Xenopus oocyte maturation is regulated by cytoplasmic polyadenylation elements within the 3'-untranslated region

The Wee1 protein tyrosine kinase is a key regulator of cell cycle progression. Wee1 activity is necessary for the control of the first embryonic cell cycle following the fertilization of meiotically mature Xenopus oocytes. Wee1 mRNA is present in immature oocytes, but Wee1 protein does not accumulate in immature oocytes or during the early stages of progesterone-stimulated maturation. This delay in Wee1 translation is critical since premature Wee1 protein accumulation has been shown to inhibit oocyte maturation. In this study we provide evidence that Wee1 protein accumulation is regulated at the level of mRNA translation. This translational control is directed by sequences within the Wee1 mRNA 3'-untranslated region (3' UTR). Specifically, cytoplasmic polyadenylation element (CPE) sequences within the Wee1 3' UTR are necessary for full translational repression in immature oocytes. Our data further indicate that while CPE-independent mechanisms may regulate the levels of Wee1 protein accumulation during progesterone-stimulated oocyte maturation, the timing of Wee1 mRNA translational induction is directed through a CPE-dependent mechanism.     

Genome level analysis of rice mRNA 3'-end processing signals and alternative polyadenylation

The position of a poly(A) site of eukaryotic mRNA is determined by sequence signals in pre-mRNA and a group of polyadenylation factors. To reveal rice poly(A) signals at a genome level, we constructed a dataset of 55 742 authenticated poly(A) sites and characterized the poly(A) signals. This resulted in identifying the typical tripartite cis-elements, including FUE, NUE and CE, as previously observed in Arabidopsis. The average size of the 3′-UTR was 289 nucleotides. When mapped to the genome, however, 15% of these poly(A) sites were found to be located in the currently annotated intergenic regions. Moreover, an extensive alternative polyadenylation profile was evident where 50% of the genes analyzed had more than one unique poly(A) site (excluding microheterogeneity sites), and 13% had four or more poly(A) sites. About 4% of the analyzed genes possessed alternative poly(A) sites at their introns, 5′-UTRs, or protein coding regions. The authenticity of these alternative poly(A) sites was partially confirmed using MPSS data. Analysis of nucleotide profile and signal patterns indicated that there may be a different set of poly(A) signals for those poly(A) sites found in the coding regions. Based on the features of rice poly(A) signals, an updated algorithm termed PASS-Rice was designed to predict poly(A) sites.     

Analysis of 3'-untranslated regions of seven c-myc genes reveals conserved elements prevalent in post-transcriptionally regulated genes

We have characterized the complete sequence of two c-myc cDNAs from the amphibian Xenopus laevis, and could thus compare the 3'-non-coding sequences of 7 myc cDNAs from 6 species spread over 350 million years of evolution. Although the size of these sequences is heterogeneous, we identified three completely conserved sequences of 10, 11 and 12 contiguous nucleotides. We observed that two of these elements may be contained in conserved stem-loop structures previously implicated in mRNA turnover. The length of these motifs, their existence in conserved predicted structures, and their presence in regulated eukaryote mRNA with a frequency greater than predicted by chance, suggest that they are functionally important.     

Acridine-linked oligonucleotide probes for the short dAT-rich motifs in the 3'-untranslated region of cytokine genes

We have investigated the use of oligonucleotide probes for identifying cDNA clones containing the short dAT-rich motifs found in the 3'-untranslated region of cytokine genes. To obtain sufficiently stable duplexes between the octameric probes used to identify genes containing the sequence dTATTTATT and its complement, it was necessary to couple an intercalating agent, an acridine derivative (acr), to the 5'-positions of the probes. The resulting octamers 5'-acr-dAATAAATA and, particularly, 5'-acr-dTATTTATT were successfully used to distinguish the complementary sequences in cDNA from internal, single point mismatched sequences. Southern blot analyses of plasmids containing IL-1 beta and IL-8 gave positive results with the 3' degenerate probe, 5'-acr-dTATTTATTN, clearly showing that the very short probe approach can be used in this type of analysis. Subsequently, in slot blot analyses we found that, even without the degenerate nucleotide, N, plasmids bearing cytokine sequences with at least 7 contiguous matched nucleotides could be unambiguously identified with 5'-acr-dTATTTATT. Unfortunately, because of the ubiquity of these dAT-rich sequences in bacterial DNA, it was not possible to use these probes for direct colony screening. In contrast to the results obtained with DNA, at the RNA level, with IL-1 beta mRNA bound to nitrocellulose, the hybrid formed with 5'-acr-dAATAAATA was very unstable, even in 1M LiCl solution at 2 degrees C; however, in the same salt solution the slightly longer acridine-coupled probes 5'-acr-dAATAAATAGGG and 5'-acr-dAAAGAACAA remained hybridized to their complementary sequences up to about 18 degrees C.     

Selection of sequence elements that substitute for the standard AATAAA motif which signals 3'' processing and polyadenylation of late simian virus 40 mRNAs.

A method is described which allows selection of sequences which can substitute for the normal AATAAA hexanucleotide involved in polyadenylation of SV40 late mRNAs. Plaques were generated from viral DNA lacking the motif, forcing acquisition of substitute sequences. Four variants were characterized. All displayed wild-type growth kinetics and produced normal levels of late mRNAs and proteins. Two variants had reacquired AATAAA elements and one acquired an ATTAAA sequence. The last variant carried an ATTTTTTAAA segment, suggesting this novel sequence, or some portion of it, can also signal poly A addition.     

Secondary structure as a functional feature in the downstream region of mammalian polyadenylation signals

Secondary structure within the downstream region of mammalian polyadenylation signals has been proposed to perform important functions. The simian virus 40 late polyadenylation signal (SVLPA) forms alternate secondary structures in equilibrium. Their formation correlates with cleavage-polyadenylation efficiency (H. Hans and J. C. Alwine, Mol. Cell. Biol. 20:2926-2932, 2000; M. I. Zarudnaya, I. M. Kolomiets, A. L. Potyahaylo, and D. M. Hovorun, Nucleic Acids Res. 3:1375-1386, 2003), and oligonucleotides that disrupt the secondary structure inhibit in vitro cleavage. To define the important features of downstream secondary structure, we first minimized the SVLPA by deletion, forming a downstream region with fewer, and more stable, stem-loop structures. Specific mutagenesis showed that both stem stability and loop size are important functional features of the downstream region. Stabilization of the stem, thus minimizing alternative structures, decreased cleavage efficiency both in vitro and in vivo. This was most deleterious when the stem was stabilized at the base of the loop, constraining loop size by inhibiting breathing of the stem. The significance of loop size was supported by mutants that showed increased cleavage efficiency with increased loop size and vice versa. A loop of at least 12 nucleotides promoted cleavage; U richness in the loop also promoted cleavage and was particularly important when the stem was stabilized. A mutation designed to eliminate downstream secondary structure still formed many relatively weak alternative structures in equilibrium and retained function. The data suggest that although the downstream region is very important, its structure is quite malleable and is able to tolerate significant mutation within a wide range of primary and secondary structural features. We propose that this malleability is due to the enhanced ability of GU- and U-rich downstream elements to easily form secondary structures with surrounding sequences.     

Upstream and downstream cis-acting elements for cleavage at the L4 polyadenylation site of adenovirus-2.

A study of the cis-acting elements involved in the 3' end formation of the RNAs from the major late L4 family of adenovirus-2 was undertaken. Series of 5' or 3' end deletion mutants and mutants harboring either internal deletions or substitutions were prepared and assayed for in vitro cleavage. This first allowed the demonstration of a sequence, located at -6 to -29, relative to AAUAAA, whose deletion or substitution reduces cleavage efficiency at the L4 polyadenylation site two to three fold. This upstream efficiency element 5' AUCUUUGUUGUC/AUCUCUGUGCUG 3' is constituted of a partially repeated 12 nucleotide long, UCG rich sequence. The activities of the 2 sequence elements in cleavage are additive. We also searched for regulatory sequences downstream of the L4 polyadenylation site. We found that the deletion or substitution of a 30 nucleotide long UCG rich sequence, between nucleotides +7 and +35 relative to the cleavage site and harboring a UCCUGU repeat reduces cleavage efficiency at least ten fold. A GUUUUU sequence, starting at +35 had no influence. Thus, the usage of the L4 polyadenylation site requires down-stream sequences different from the canonical GU or U boxes and is regulated by upstream sequence elements.     

Mutated polyadenylation signals for controlling expression levels of multiple genes in mammalian cells

A set of mutated SV40 early polyadenylation signals (SV40pA) with varying strengths is generated by mutating the AATAAA sequence in the wild-type SV40pA. They are shown to control the expression level of a gene over a 10-fold range using luciferase reporter genes in transient transfection assays. The relative strength of these SV40pA variants remains similar under three commonly used mammalian promoters and in five mammalian cell lines. Application of SV40pA variants for controlling expression level of multiple genes is demonstrated in a study of monoclonal antibody (mAb) synthesis in mammalian cells. By using SV40pA variants of different strengths, the expression of light chain (LC) and heavy chain (HC) genes encoded in a single vector is independently altered which results in different ratios of LC to HC expression spanning a range from 0.24 to 16.42. The changes in gene expression are determined by measuring mRNA levels and intracellular LC and HC polypeptides. It is found that a substantial decrease of HC expression, which increases the LC/HC mRNA ratio, only slightly reduces mAb production. However, reducing the LC expression by a similar magnitude, which decreases the LC/HC mRNA ratio results in a sharp decline of mAb production to trace amounts. This set of SV40pA variants offers a new tool for accurate control of the relative expression levels of multiple genes. It will have wide-ranging applications in fields related to the study of biosynthesis of multi-subunit proteins, proteomic research on protein interactions, and multi-gene metabolic engineering.