U2

Pre-mRNA

▲ FIGURE 12-12 Schematic diagrams comparing the secondary structures of group II self-splicing introns (a) and U snRNAs present in the spliceosome (b). The first transesterification reaction is indicated by green arrows; the second reaction, by blue arrows. The branch-point A is boldfaced. The similarity in these structures suggests that the spliceosomal snRNAs evolved from group II introns, with the trans-acting snRNAs being functionally analogous to the corresponding domains in group II introns. The colored bars flanking the introns in (a) and (b) represent exons [Adapted from P A. Sharp, 1991, Science 254:663.]

suggests that the splicing reaction is catalyzed by the snRNA, not the protein, components of spliceosomes. Although group II introns can self-splice in vitro at elevated temperatures and Mg2+ concentrations, under in vivo conditions proteins called maturases, which bind to group II intron RNA, are required for rapid splicing. Maturases are thought to stabilize the precise three-dimensional interactions of the intron RNA required to catalyze the two splicing transesterification reactions. By analogy, snRNP proteins in spliceosomes are

thought to stabilize the precise geometry of snRNAs and in-tron nucleotides required to catalyze pre-mRNA splicing.

The evolution of snRNAs may have been an important step in the rapid evolution of higher eukaryotes. As internal intron sequences were lost and their functions in RNA splicing supplanted by trans-acting snRNAs, the remaining intron sequences would be free to diverge. This in turn likely facilitated the evolution of new genes through exon shuffling since there are few constraints on the sequence of new introns generated in the process (see Figure 10-17). It also permitted the increase in protein diversity that results from alternative RNA splicing and an additional level of gene control resulting from regulated RNA splicing.

Most Transcription and RNA Processing Occur in a Limited Number of Domains in Mammalian Cell Nuclei

Several kinds of studies suggest that transcription and RNA processing occur largely within discrete foci within the nucleus of eukaryotic cells. For instance, digital imaging microscopy of human fibroblasts reveals that most of the nuclear polyadenylated RNA (i.e., unspliced and partially spliced pre-mRNA and nuclear mRNA) is localized in about 100 foci (Figure 12-13). An SR protein involved in exon definition (SC-35) is localized to the center of these same loci.

These data imply that the nucleus is not simply an unorganized container for chromatin and the proteins that carry out chromosome replication, transcription, and RNA processing. Rather, there is a highly organized underlying nuclear substructure. A poorly understood fibrillar network comprising multiple proteins called the nuclear matrix can be oo <1

▲ EXPERIMENTAL FIGURE 12-13 Staining shows that polyadenylated RNA and RNA splicing factors are sequestered in discrete areas of the mammalian fibroblast nucleus. Digital Imaging microscopy was used to reconstruct a 1-^m-thick section of a stained human fibroblast nucleus. (a) Section stained with red rhodaminelabeled poly(dT) to detect polyadenylated RNA (red) and with DAPI to detect DNA (blue). Polyadenylated RNA is localized to a limited number of discrete foci (speckles) between regions of chromatin, although not all regions containing low levels of DNA contained detectable polyadenylated RNA (arrow). (b) The same section shown in (a) stained to detect polyadenylated RNA (red) and SR protein SC-35, which was visualized with a green fluorescein-labeled monoclonal antibody. Regions where the stains overlap appear yellow. SC-35 is present in the center of many foci (arrow). Nu = nucleolus. [From K. C. Carter et al., 1993, Science 259:1330.]

observed in the nucleus after digestion of most DNA and extraction with high salt. This network of fibrous proteins probably contributes to nuclear organization just as cy-toskeletal fibers contribute to the organization of the cytoplasm. Much remains to be learned about the molecular mechanisms underlying this organization and its consequences for nuclear processes.

Nuclear Exonucleases Degrade RNA That Is Processed out of Pre-mRNAs

Because the human genome contains long introns, only =5 percent of the nucleotides that are polymerized by RNA poly-merase II during transcription are retained in the mature, processed mRNA. The introns that are spliced out and the region downstream from the cleavage and polyadenylation site are degraded by nuclear exonucleases that hydrolyze one base at a time from either the 5' or 3' end of an RNA strand.

As mentioned earlier, the 2',5'-phosphodiester bond in excised introns is hydrolyzed by a debranching enzyme, yielding a linear molecule with unprotected ends that can be attacked by exonucleases (see Figure 12-9). The predominant nuclear decay pathway is 3'^5' hydrolysis by 11 exonucleases that associate with one another in a large protein complex called the exosome. Other proteins in the complex include RNA heli-cases that disrupt base pairing and RNA-protein interactions that would otherwise impede the exonucleases. Exosomes also function in the cytoplasm as discussed later. In addition the ex-osome appears to degrade pre-mRNAs that have not been properly spliced or polyadenylated. It is not yet clear how the exosome recognizes improperly processed pre-mRNAs.

To avoid being degraded by nuclear exonucleases, nascent transcripts, pre-mRNA processing intermediates, and mature mRNAs in the nucleus must have their ends protected. The 5' end of a nascent transcript is protected by addition of the 5' cap structure as soon as the 5' end emerges from the poly-merase. Further protection is provided by interaction of the cap with a nuclear cap-binding complex, which also functions in export of mRNA to the cytoplasm. The 3' end of a nascent transcript lies within the RNA polymerase and thus is inaccessible to exonucleases (see Figure 4-10). As discussed previously, the free 3' end generated by cleavage of a pre-mRNA downstream from the poly(A) signal is rapidly polyadenylated by the poly(A) polymerase associated with the other 3' processing factors, and the resulting poly(A) tail is bound by PABPII (see Figure 12-4). This tight coupling of cleavage and polyadenylation protects the 3' end from exonuclease attack.

■ Shortly after transcription initiation, a capping enzyme associated with the phosphorylated CTD of RNA poly-merase II adds the 5' cap to the nascent transcript.

■ In most protein-coding genes, a conserved AAUAAA poly (A) signal lies slightly upstream from a poly (A) site where cleavage and polyadenylation occur. A GU- or U-rich sequence downstream from the poly(A) site contributes to the efficiency of cleavage and polyadenylation.

■ A multiprotein complex that includes poly(A) poly-merase (PAP) carries out the cleavage and polyadenylation of a pre-mRNA. A nuclear poly(A)-binding protein, PABPII, stimulates addition of A residues by PAP and stops addition once the poly (A) tail reaches 200-250 residues (see Figure 12-4).

■ Five different snRNPs interact via base pairing with one another and with pre-mRNA to form the spliceosome (see Figure 12-9). This very large ribonucleoprotein complex catalyzes two transesterification reactions that join two ex-ons and remove the intron as a lariat structure, which is subsequently degraded (see Figure 12-7).

■ The association of pre-mRNA processing factors with the CTD of RNA polymerase II stimulates chain elongation. This coupling ensures that a pre-mRNA is not synthesized until the factors required for its processing are in place to interact with splicing and cleavage and polyadenyl-ation signals in the pre-mRNA as they emerge from the polymerase.

■ SR proteins that bind to exonic splicing enhancer sequences in exons are critical in defining exons in the large pre-mRNAs of higher organisms. A network of interactions between SR proteins, snRNPs, and splicing factors forms a cross-exon recognition complex that specifies correct splice sites (see Figure 12-11).

■ The snRNAs in the spliceosome are thought to have an overall tertiary structure similar to that of group II self-splicing introns.

■ For long transcription units in higher organisms, splicing of exons usually begins as the pre-mRNA is still being formed. Cleavage and polyadenylation to form the 3' end of the mRNA occur after the poly(A) site is transcribed.

■ Excised introns are degraded primarily by exosomes, multiprotein complexes that contain eleven 3'^5' exonu-cleases as well as RNA helicases. Exosomes also degrade improperly processed pre-mRNAs.

Lower Your Cholesterol In Just 33 Days

Lower Your Cholesterol In Just 33 Days

Discover secrets, myths, truths, lies and strategies for dealing effectively with cholesterol, now and forever! Uncover techniques, remedies and alternative for lowering your cholesterol quickly and significantly in just ONE MONTH! Find insights into the screenings, meanings and numbers involved in lowering cholesterol and the implications, consideration it has for your lifestyle and future!

Get My Free Ebook


Post a comment