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Riddle DL, Blumenthal T, Meyer BJ, et al., editors. C. elegans II. 2nd edition. Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press; 1997.

Cover of C. elegans II

C. elegans II. 2nd edition.

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Section VIII3′-End Formation

In vertebrates, formation of 3′ends of mRNAs is dependent on a complex of proteins binding to signals just upstream of and just downstream from the future cleavage site, followed by cleavage of the pre-mRNA and polyadenylation of the resulting free 3′end (for review, see Wahle and Keller 1992). The upstream site is the almost perfectly conserved sequence, AAUAAA, which occurs between 10 and 35 nucleotides upstream of the cleavage site. This site binds a complex of three polypeptides called CPSF. The downstream site is a much less highly conserved U-rich or GU-rich sequence that binds another three-subunit protein called CStF. In addition, other proteins are required for the cleavage and polyadenylation reactions.

It appears that signals for 3′-end formation have diverged more rapidly than have signals for splicing. Although these signals have been very well defined in vertebrates, it has not proved possible to use this information to identify 3′-end formation signals in yeast or plants. In contrast, many C. elegans genes do have the AAUAAA sequence just upstream of the site of poly(A) addition. On the other hand, many do not. In an effort to determine what the 3′-end formation signal is in C. elegans, we have surveyed the 3′ends of published genes and cDNAs for matches to the AAUAAA consensus (Table 3 and Fig. 8). In addition, an analysis of 3′-end sequences from a random set of 1500 C. elegans cDNA clones came to very similar conclusions (T. Blumenthal et al., unpubl.). These surveys clearly show that C. elegans uses the same signal as vertebrates, but the sequence requirements are less stringent. Just over half of C. elegans genes have a perfect match to the consensus AAUAAA. Most of the remaining genes have one of a limited group of tolerated mismatches to the consensus. AAUGAA (11%) and UAUAAA (8%) are the most commonly used variants. Furthermore, AAUAAA and variants occur in a very limited region, generally 11−17 nucleotides upstream of the cleavage site. Interestingly, 7% of C. elegans genes do not have any sequence related to AAUAAA in this region. This suggests that C. elegans may have an alternative, but much less commonly used, mechanism for 3′-end formation.

Table 3. Polyadenylation and cleavage signal.

Table 3

Polyadenylation and cleavage signal.

Figure 8. Distance between the AAUAAA and the poly(A) site.

Figure 8

Distance between the AAUAAA and the poly(A) site. Each bar represents the number of genes with the precise number of base pairs between the 3′ end of the AAUAAA and the cleavage site indicated below. (more...)

Because the C. elegans 3′-end formation machinery can accurately locate the correct cleavage site with several variants of the AAUAAA sequence, it is reasonable to suppose that the downstream signal might have a more highly conserved sequence than that in vertebrates. However, there is no obvious U-rich or GU-rich element in the downstream region. Furthermore, a search for an alternative consensus sequence in this region has not revealed any candidates. Additional sequences needed for 3′-end formation in C. elegans await identification.

One alternative mechanism for 3′-end formation that does appear to occur in C. elegans is polyadenylation at the free 3′end created by trans- splicing. Spieth et al. (1993) isolated cDNA clones in which the mai-1 mRNA was polyadenylated at, or just a base or two upstream of, the gpd-2 trans-splice site, instead of the normal site 100 nucleotides upstream. They hypothesized that these arose by polyadenylation of the free 3′end created by trans-splicing. This might require that the intercistronic region be debranched, and it would require that the mai-1 AAUAAA sequence 100 nucleotides upstream serve as a binding site for CPSF to allow polyadenylation at a distance. This sequence has been shown to be able to function at a distance in vertebrates (Manley et al. 1985). The mai-1 sequence, AGUAAA, is used very rarely, which may explain why a portion of the mRNA does not form 3′ends in the normal way. This alternative mechanism for 3′-end formation is probably used as well in the rare operons in which the site of polyadenylation of the upstream gene and the trans-splice site of the downstream gene are the same (Hengartner and Horvitz 1994b; I. Korf and S. Strome, pers. comm.). Presumably, the mature mRNAs are formed by internal trans-splicing by the SL1 snRNP, followed by polyadenylation of the upstream mRNA at the free 3′end created by trans-splicing. Again, the upstream mRNA might have to be debranched, but in these cases, there is a proximal AAUAAA for binding CPSF to catalyze polyadenylation. It should be mentioned that, in general, 3′ends of genes within operons are signaled by AAUAAA and that the degree of conservation of this sequence at these locations is similar to that shown in Table 3.

Copyright © 1997, Cold Spring Harbor Laboratory Press.
Bookshelf ID: NBK20220
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