Several distinct types of sequence elements are required for efficient mRNA 3'' end formation in a pea rbcS gene.

We have conducted an extensive linker substitution analysis of the polyadenylation signal from a pea rbcS gene. From these studies, we can identify at least two, and perhaps three, distinct classes of cis element involved in mRNA 3' end formation in this gene. One of these, termed the far-upstream element, is located between 60 and 120 nt upstream from its associated polyadenylation sites and appears to be largely composed of a series of UG motifs. A second, termed the near-upstream element, is more proximate to poly(A) sites and may be functionally analogous to the mammalian polyadenylation signal AAUAAA, even though the actual sequences involved may not be AAUAAA. The third possible class is the putative cleavage and polyadenylation site itself. We find that the rbcS-E9 far-upstream element can replace the analogous element in another plant polyadenylation signal, that from cauliflower mosaic virus, and that one near-upstream element can function with either of two poly(A) sites. Thus, these different cis elements are largely interchangeable. Our studies indicate that a cellular plant gene possesses upstream elements distinct from AAUAAA that are involved in mRNA 3' end formation and that plant genes probably have modular, multicomponent polyadenylation signals.     

Different sequence elements are required for function of the cauliflower mosaic virus polyadenylation site in Saccharomyces cerevisiae compared with in plants.

We show that the polyadenylation site derived from the plant cauliflower mosaic virus (CaMV) is specifically functional in the yeast Saccharomyces cerevisiae. The mRNA 3' endpoints were mapped at the same position in yeast cells as in plants, and the CaMV polyadenylation site was recognized in an orientation-dependent manner. Mutational analysis of the CaMV 3'-end-formation signal revealed that multiple elements are essential for proper activity in yeast cells, including two upstream elements that are situated more than 100 and 43 to 51 nucleotides upstream of the poly(A) addition site and the sequences at or near the poly(A) addition site. A comparison of the sequence elements that are essential for proper function of the CaMV signal in yeast cells and plants showed that both organisms require a distal and a proximal upstream element but that these sequence elements are not identical in yeast cells and plants. The key element for functioning of the CaMV signal in yeast cells is the sequence TAGTATGTA, which is similar to a sequence previously proposed to act in yeast cells as a bipartite signal, namely, TAG ... TATGTA. Deletion of this sequence in the CaMV polyadenylation signal abolished 3'-end formation in yeast cells, and a single point mutation in this motif reduced the activity of the CaMV signal to below 15%. These results indicate that the bipartite sequence element acts as a signal for 3'-end formation in yeast cells but only together with other cis-acting elements.     

The contribution of AAUAAA and the upstream element UUUGUA to the efficiency of mRNA 3''-end formation in plants.

The requirement for sequence specificity in the AAUAAA motif of the cauliflower mosaic virus (CaMV) polyadenylation signal was examined by saturation mutagenesis. While deletion of AAUAAA almost abolished processing at the CaMV polyadenylation site, none of the 18 possible single base mutations had a dramatic effect on processing efficiency. The effect of replacing all six nucleotides simultaneously varied depending on the sequence used, but some replacements were as detrimental as the deletion mutant. Taken together, these results confirm that AAUAAA is an essential component of the CaMV polyadenylation signal, but indicate that a high degree of sequence variation can be tolerated. A repeated UUUGUA motif was identified as an important upstream accessory element of the CaMV polyadenylation signal. This sequence was able to induce processing at a heterologous polyadenylation site in a sequence-specific and additive manner. The effect of altering the spacing between this upstream element and the AAUAAA was examined; moving these two elements closer together or further apart reduces the processing efficiency. The upstream element does not function to signal processing at the CaMV polyadenylation site if placed downstream of the cleavage site. Analysis of further upstream sequences revealed that almost all of the 200 nt fragment required for maximal processing contributes positively to processing efficiency. Furthermore, isolated far upstream sequences distinct from UUUGUA were also able to induce processing at a heterologous polyadenylation site.     

Elements upstream of the AAUAAA within the human immunodeficiency virus polyadenylation signal are required for efficient polyadenylation in vitro.

Recent in vivo studies have identified specific sequences between 56 and 93 nucleotides upstream of a polyadenylation [poly(A)] consensus sequence, AAUAAA, in human immunodeficiency virus type 1 (HIV-1) that affect the efficiency of 3'-end processing at this site (A. Valsamakis, S. Zeichner, S. Carswell, and J. C. Alwine, Proc. Natl. Acad. Sci. USA 88:2108-2112, 1991). We have used HeLa cell nuclear extracts and precursor RNAs bearing the HIV-1 poly(A) signal to study the role of upstream sequences in vitro. Precursor RNAs containing the HIV-1 AAUAAA and necessary upstream (U3 region) and downstream (U5 region) sequences directed accurate cleavage and polyadenylation in vitro. The in vitro requirement for upstream sequences was demonstrated by using deletion and linker substitution mutations. The data showed that sequences between 56 and 93 nucleotides upstream of AAUAAA, which were required for efficient polyadenylation in vivo, were also required for efficient cleavage and polyadenylation in vitro. This is the first demonstration of the function of upstream sequences in vitro. Previous in vivo studies suggested that efficient polyadenylation at the HIV-1 poly(A) signal requires a spacing of at least 250 nucleotides between the 5' cap site and the AAUAAA. Our in vitro analyses indicated that a precursor containing the defined upstream and downstream sequences was efficiently cleaved at the polyadenylation site when the distance between the 5' cap and the AAUAAA was reduced to at least 140 nucleotides, which is less than the distance predicted from in vivo studies. This cleavage was dependent on the presence of the upstream element.     

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.     

3'-end processing of the maize 27 kDa zein mRNA

Cis -regulatory elements involved in the mRNA 3'-end processing of the 27 kDa zein gene have been investigated by deletion and site-directed mutagenesis analyses. In the 3' flanking region of the 27 kDa zein gene, several AATAAA-like sequences and a sequence resembling the mammalian GT-rich sequence are present around the polyadenylation sites. Among the multiple AATAAA-like sequences, the duplicated AATGAA motifs, located 30–40 bp upstream from the polyadenylation sites, have been shown to play roles as polyadenylation signals. Although either of the two AATGAA motifs can function as a polyadenylation signal in chimeric gene constructs, the one proximal to the polyadenylation sites is likely to be the functional polyadenylation signal in the 27 kDa zein gene. Deletion of the downstream GT-rich sequence as well as alteration of the sequence surrounding the poly-adenylation sites has little effect on the mRNA 3'-end processing. However, the sequence elements located upstream from the polyadenylation signals are essential for the mRNA 3'-end processing. Mutations in the AATGAA motifs or the upstream sequences reduced the level of a reporter gene expression. A model depicting the mechanism involved in the 3'-end processing of the 27 kDa zein mRNA is presented.     

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.     

Spatial constraints on polyadenylation signal function

Efficient cleavage and polyadenylation of eukaryotic messenger RNAs require at least two signal elements: an AAUAAA or closely related sequence located 7-30 base pairs (bp) upstream of the site of processing, and a G/U- or U-rich sequence located 3' to the cleavage site. The herpes simplex virus type 1 thymidine kinase (tk) gene contains two copies of the AATAAA hexanucleotide and a GT-rich region. We have shown that the first AATAAA and the GT-rich region are essential for efficient processing, both in vivo and in vitro, whereas the second AATAAA does not appear to play any role in the formation of tk mRNA 3' ends. The failure of a signal containing only the second AATAAA and the GT-rich element to signal cleavage and polyadenylation suggested that these two elements might be too close together to constitute a functional polyadenylation signal. The experiments described in this report were directed at determining the effects on mRNA 3' end formation of alterations in spacing between signal elements. Wild-type tk contains 19 bp between these two elements. Constructs were made in which an AATAAA and the GT-rich region were separated by various distances ranging from 7 to 43 bp. The quantity and location of 3' ends of the tk mRNA produced by these constructs in Cos-1 cells were measured by S1 nuclease protection analysis. Signal efficiency was gradually reduced as the separation between the two signal elements was increased; with a separation of 43 bp, the signal functioned at approximately one-eighth the efficiency of the parental construction. Bringing the two signals closer together resulted in decreased signal efficiency; with a separation of 7 or 9 bp, no tk mRNA polyadenylated within the normal region was produced. Altering the sequences between these two elements without changing the distance had small effects on processing efficiency.     

Upstream elements present in the 3'-untranslated region of collagen genes influence the processing efficiency of overlapping polyadenylation signals

3'-Untranslated regions (UTRs) of genes often contain key regulatory elements involved in gene expression control. A high degree of evolutionary conservation in regions of the 3'-UTR suggests important, conserved elements. In particular, we are interested in those elements involved in regulation of 3' end formation. In addition to canonical sequence elements, auxiliary sequences likely play an important role in determining the polyadenylation efficiency of mammalian pre-mRNAs. We identified highly conserved sequence elements upstream of the AAUAAA in three human collagen genes, COL1A1, COL1A2, and COL2A1, and demonstrate that these upstream sequence elements (USEs) influence polyadenylation efficiency. Mutation of the USEs decreases polyadenylation efficiency both in vitro and in vivo, and inclusion of competitor oligoribonucleotides representing the USEs specifically inhibit polyadenylation. We have also shown that insertion of a USE into a weak polyadenylation signal can enhance 3' end formation. Close inspection of the COL1A2 3'-UTR reveals an unusual feature of two closely spaced, competing polyadenylation signals. Taken together, these data demonstrate that USEs are important auxiliary polyadenylation elements in mammalian genes.     

Cleavage and polyadenylation of messenger RNA precursors in vitro occurs within large and specific 3' processing complexes.

We have investigated the assembly of complexes associated with in vitro cleavage and polyadenylation of synthetic pre-mRNAs by native gel electrophoresis. Incubation of SP6-generated pre-mRNA containing the adenovirus L3 polyadenylation site in HeLa cell nuclear extract results in the rapid assembly of specific complexes. Formation of these complexes precedes the appearance of cleaved intermediates and polyadenylated products and is dependent on an intact polyadenylation signal within the pre-mRNA. The specific complexes do not form on RNAs with point mutations in the AAUAAA sequence upstream of the L3 polyadenylation site. Furthermore, such mutant RNAs cannot compete for factors involved in the assembly of specific complexes on wild-type pre-mRNA. Upon complex formation a 67-nucleotide region of the L3 pre-mRNA is protected from RNase T1 digestion. This region contains both the upstream AAUAAA signal and the GU-rich downstream sequences. Cleavage and polyadenylation occur within the specific complexes and the processed RNA is subsequently released. We propose that the assembly of specific complexes represents an essential step during pre-mRNA 3' end formation in vitro.