MCB 201 Gene Expression - Spring Semester 2003
Lecture 20 (chapter 11 RNA Processing, Nuclear Transport, and Post-transcriptional Control cont.)
Section 11.2: Processing of Eukaryotic mRNA cont.
1. Figure 11-12: Model for cleavage and polyadenylation of pre-mRNAs in mammalian cells. In animal cells, all mRNAs except the histone mRNAs have the sequence AAUAAA located about 10-35 nucleotides upstream from the poly(A) tail. This is the site in the primary RNA transcript where a protein complex called cleavage and polyadenylation specificity factor (CPSF) binds. Other proteins also bind to this same site to build up the complex further. Another protein called cleavage stimulatory factor (CStF) binds to a nearby G/U rich sequence. These two complexes then interact with each other forming a loop in the RNA, a structure that is further stabilized by the addition of other proteins. The purpose of this complex is to create a binding site for the poly(A) polymerase (PAP). The next key event is the binding of PAP, which binds before cleavage occurs. This allows the process of cleavage of the RNA to be linked to polyadenylation. As soon as a 3'-OH is created by cleavage of a phosphodiester bond, PAP begins adding A's. The first approximately 12 A residues are added slowly. These residues become a binding site for poly(A)binding protein II (PABII). This protein interacts with PAP to speed up addition of another 200 or so A residues, after which PABII signals PAP to stop. How PABII makes the decision to signal stop is not clear.
2. Figure 11-13: The first evidence for splicing out of introns is shown here. A piece of adenoviral DNA containing a gene with exons and introns is shown in Panel A. In Panel B, an electron micrograph of an RNA-DNA hybrid molecule is shown. This molecule was produced by allowing the piece of DNA shown in A (EcoRI A fragment) to hybridize with mature mRNA transcribed from this gene. The striking result is that intron segments are looped out as single-stranded DNA. That is, they are not represented by complementary sequences in the mature mRNA. If the hybrid is formed using primary transcript RNA instead of mature mRNA, these loops are not present and all of the sequences are base-paired, indicating that both exons and introns are present in the primary transcript.
3. Figure 11-14: How does splicing work? First, let's examine a sample of pre-mRNA, the substrate for splicing. There is very little in the way of a concensus sequence at the splice sites. Within the intron to be removed, there is a 5'-GU and a 3' AG. Also, there is an invariant A residue 20-50 bases from the 3' splice site that becomes the branch point during splicing and a short pyrimidine-rich (T, C) region further down the intron.
4. Figure 11-15: Analysis of RNA products formed in an in vitro splicing reaction. All of the protein components needed to carry out splicing are in a nuclear extract of HeLa cells. Typically ATP must be added to these reactions as an energy source. The endogenous ATP in these extracts, i.e. ATP that was in the cell before the extract was made, is hydrolyzed during extract preparation and storage. To this extract is added a test piece of RNA containing two exons separated by an intron. This RNA substrate for splicing is radioactively labeled so that we can follow its fate. The interesting thing that the separation of the reaction products on gels shows is that several of the RNA pieces have two forms, one migrating slower (marked by a single asterisk) than the other. This slower migrating RNA form was later identified as a lariat structure, which is the form of the excised intron.
5. Figure 11-16: Splicing of exons in pre-mRNA occurs via two transesterification reactions. The splicing reaction, minus the proteins that actually do the work, is shown here. It consists of two successive transesterification reactions, the net result of which is that two exons are ligated together and the intervening intron is released as a branched lariat structure, all with very little energy expenditure.
6. Figure 11-17: Diagram of interactions between pre-mRNA and several snRNAs early in the splicing process. Here again, the proteins have been omitted so that the RNA interactions can be seen more clearly. U1 snRNA has sequences complementary to the splice junction at the end of exon 1 and this region bridges the splice site. U2 snRNA has a sequence complementary to the sequence in the pre-mRNA around the branchpoint A residue, but does not include this residue, which is then readily available for the transesterification reaction. Note that human antisera from patients with the autoimmune disease systemic lupus erythematosus have antibodies against snRNP proteins, which have been very useful in characterizing them.
7. Figure 11-18: Electron micrograph of a spliceosome. These assemblies of smaller ribonucleoprotein particles (RNPs) are about the size of a ribosome.
8. Figure 11-19: The spliceosomal splicing cycle (media connections: mRNA Splicing). How does one approach a complex assembly and reaction sequence as shown here? First look for the major themes. For example, it is clear that the binding of snRNPs to the pre-mRNA substrate is ordered and that these snRNPs are recycled through multiple rounds of splicing. Notice that the spliceosome goes through at least one major reorganization which requires energy provided by ATP hydrolysis prior to the two transesterification reactions that create the lariat intron. In higher eukaryotes, a protein called U2AF binds to the pyrimidine-rich region and assists the binding of the U2 snRNP. Shortly after its release, the lariat intron is rapidly degraded.
9. Figure 11-20: Schematic diagrams comparing the secondary structures of (a) group II self-splicing introns and (b) U snRNAs present in the spliceosome. A recent unexpected finding is that some introns are self-splicing, meaning that no other proteins or RNA molecules are needed, at least in test tube (in vitro) reactions. Group I introns found in nuclear ribosomal RNA genes of protozoans and Group II introns found in mitochondria and chloroplasts of plants and fungi include self-splicing introns. As shown here, group II introns fold into a complex array of stem-loops, and these structures are evolutionarily highly conserved, even though the nucleotide sequences of these introns are not highly conserved. The self-splicing reaction involves two transesterifications and is very similar to splicing by spliceosomes. It has been suggested that the group II intron is more similar to the ancestral intron and that during evolution some groups of introns either lost stem loops or these structures took on an independent existence as snRNPs. Note the assemblage of snRNPs in panel B.
There are several other very interesting properties of group II introns. They encode maturase proteins that participate in the splicing reaction in cells, presumeably by binding to the intron and stabilizing it in a configuration that accelerates splicing. These same maturases confer another remarkable property to group II introns, allowing them to behave like mobile DNA elements in the genome. The maturase has a domain that has reverse transcriptase activity, allowing the RNA to be copied back into DNA and inserted in another location in the genome. Transposition is a rare event in general, and when group II introns move into a gene, this gene is not inactivated because the intron can self-splice out of the transcribed pre-mRNA.
10. Figure 11-21: Localization of polyadenylated RNA and RNA splicing factors in the nucleus of a mammalian fibroblast. Regions of DNA concentration are shown in blue and regions containing polyadenylated RNA, as detected by a rhodamine tagged poly-dT probe, are shown in red. Note that the polyA-RNA is located between regions of DNA staining, i.e. in their own area of the nucleus. In Panel B, the same polyA-containing RNA regions are shown along with green staining regions where the essential RNA-splicing protein SC-35 accumulated. The yellow color is regions of overlap between SC-35 and the polyA-RNA. These are probably regions where splicing occurs.
11. Figure 11-22: Transmission electron micrograph showing the nuclear matrix (skeleton) of a HeLa cell. snRNps remain associated with the nuclear skeleton shown here in detergent-treated cells that have also been digested with DNase to remove DNA and extracted with ammonium sulfate to remove histones and other chromosomal proteins. If ATP is added to these nuclear skeletons, pre-mRNAs left associated as well undergo splicing. Therefore, the localized complexes shown in figure 11-21 may be associated with nucleoskeleton, or nuclear matrix.