Harpers

354 / CHAPTER 37is accomplished by a nucleophilic attack by an adenylylresidue in the branch point sequence located just upstreamfrom the 3′ end of this intron. The free 5′ terminalthen forms a loop or lariat structure that is linkedby an unusual 5′–2′ phosphodiester bond to the reactiveA in the PyNPyPyPuAPy branch site sequence(Figure 37–12). This adenylyl residue is typically located28–37 nucleotides upstream from the 3′ end ofthe intron being removed. The branch site identifiesthe 3′ splice site. A second cut is made at the junctionof the intron with the 3′ exon (donor on right). In thissecond transesterification reaction, the 3′ hydroxyl ofthe upstream exon attacks the 5′ phosphate at thedownstream exon-intron boundary, and the lariatstructure containing the intron is released and hydrolyzed.The 5′ and 3′ exons are ligated to form a continuoussequence.The snRNAs and associated proteins are requiredfor formation of the various structures and intermediates.U1 within the snRNP complex binds first by basepairing to the 5′ exon-intron boundary. U2 within thesnRNP complex then binds by base pairing to thebranch site, and this exposes the nucleophilic A residue.U5/U4/U6 within the snRNP complex mediates anATP-dependent protein-mediated unwinding that resultsin disruption of the base-paired U4-U6 complexwith the release of U4. U6 is then able to interact firstwith U2, then with U1. These interactions serve to approximatethe 5′ splice site, the branch point with itsreactive A, and the 3′ splice site. This alignment is enhancedby U5. This process also results in the formationof the loop or lariat structure. The two ends arecleaved, probably by the U2-U6 within the snRNPcomplex. U6 is certainly essential, since yeasts deficientin this snRNA are not viable. It is important to notethat RNA serves as the catalytic agent. This sequence isthen repeated in genes containing multiple introns. Insuch cases, a definite pattern is followed for each gene,and the introns are not necessarily removed in sequence—1,then 2, then 3, etc.The relationship between hnRNA and the correspondingmature mRNA in eukaryotic cells is now apparent.The hnRNA molecules are the primary transcriptsplus their early processed products, which, afterthe addition of caps and poly(A) tails and removal ofthe portion corresponding to the introns, are transportedto the cytoplasm as mature mRNA molecules.Alternative Splicing Providesfor Different mRNAsThe processing of hnRNA molecules is a site for regulationof gene expression. Alternative patterns ofRNA splicing result from tissue-specific adaptive anddevelopmental control mechanisms. As mentionedabove, the sequence of exon-intron splicing events generallyfollows a hierarchical order for a given gene. Thefact that very complex RNA structures are formed duringsplicing—and that a number of snRNAs and proteinsare involved—affords numerous possibilities for achange of this order and for the generation of differentmRNAs. Similarly, the use of alternative terminationcleavage-polyadenylationsites also results in mRNAheterogeneity. Some schematic examples of theseprocesses, all of which occur in nature, are shown inFigure 37–13.Faulty splicing can cause disease. At least oneform of β-thalassemia, a disease in which the β-globingene of hemoglobin is severely underexpressed, appearsto result from a nucleotide change at an exon-intronjunction, precluding removal of the intron and thereforeleading to diminished or absent synthesis of theβ-chain protein. This is a consequence of the fact thatthe normal translation reading frame of the mRNA isdisrupted—a defect in this fundamental process (splicing)that underscores the accuracy which the process ofRNA-RNA splicing must achieve.Alternative Promoter UtilizationProvides a Form of RegulationTissue-specific regulation of gene expression can beprovided by control elements in the promoter or by themRNA precursor1Selective splicing1Alternative 5′ donor site1′Alternative 3′ acceptor site112 3 AAUAA AAUAA (A) n2 3 AAUAA AAUAA (A) n222′Alternative polyadenylation site3 AAUAA AAUAA (A) n3 AAUAA AAUAA (A) n3 AAUAA (A) nFigure 37–13. Mechanisms of alternative processingof mRNA precursors. This form of RNA processinginvolves the selective inclusion or exclusion of exons,the use of alternative 5′ donor or 3′ acceptor sites, andthe use of different polyadenylation sites.