Molecular Biology Fifth Edition Lecture PowerPoint to accompany Molecular Biology Fifth Edition Robert F. Weaver Chapter 14 RNA Processing I: Splicing Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

mRNA Processing Events Most eukaryotic genes, in contrast to typical bacterial genes, are interrupted by noncoding DNA RNA polymerases cannot distinguish the noncoding regions from the coding regions, so it transcribes everything The cell must remove the noncoding RNA from the primary transcript via splicing Eukaryotes also add special structures to the 5’ and 3’ ends of the transcript, called the cap and poly-A tail, respectively All events occur in the nucleus before the mRNA emigrates to the cytoplasm

This is bhgty the human b-globin qwtzptlrbn gene. 14.1 Genes in Pieces Consider the sequence of the human b-globin gene as a sentence: This is bhgty the human b-globin qwtzptlrbn gene. Two italicized regions make no sense Contain sequences unrelated to the globin coding sequences surrounding them Intervening sequences, IVSs, or introns Parts of the gene making sense Coding regions or exons Some lower eukaryotic genes have no introns

Evidence for Split Genes Most higher eukaryotic genes coding for mRNA, tRNA and a few coding for rRNA are interrupted by unrelated regions called introns Other parts of the gene, surrounding the introns, are called exons Exons contain the sequences that finally appear in the mature RNA product Genes for mRNAs have been found with anywhere from 0 to 362 introns tRNA genes have either 0 or 1 intron

RNA Splicing Introns are present in genes but not in mature RNA How does the information not find its way into mature RNA products of the genes? Possibility 1: Introns are never transcribed Polymerase somehow jumps from one exon to another Possibility 2: Introns are transcribed Primary transcript result, an overlarge gene product is cut down by removing introns This is correct process The process of cutting introns out of immature RNAs and stitching together the exons to form the final product is RNA splicing

Splicing Outline Introns are transcribed along with exons in the primary transcript Introns are removed as the exons are spliced together

Stages of RNA Splicing Messenger RNA synthesis in eukaryotes occurs in stages First stage: Synthesis of primary transcript product This is an mRNA precursor containing introns copied from the gene if present Precursor is part of a pool of heterogeneous nuclear RNAs – hnRNAs Second stage: mRNA maturation Removal of introns in a process called splicing

Splicing Signals Splicing must be precise Splicing signals in nuclear mRNA precursors are remarkably uniform First 2 bases of introns are GU Last 2 are AG 5’- and 3’-splice sites have consensus sequences extending beyond GU and AG motifs Whole consensus sequences are important to proper splicing Abnormal splicing can occur when the consensus sequences are mutated

14.2 Mechanism of Splicing of Nuclear mRNA Precursors Intermediate in nuclear mRNA precursor splicing is branched – looks like a lariat Two-step model 2’-OH group of adenosine nucleotide in middle of intron attacks phosphodiester bond between 1st exon and G beginning of intron Forms loop of the lariat Separates first exon from intron 3’-OH left at end of 1st exon attacks phosphodiester bond linking intron to 2nd exon Forms the exon-exon phosphodiester bond Releases intron in lariat form at same time

Simplified Mechanism of Splicing 2’-OH group of A within intron attackes the phosphodiester bond linking the first exon to the intron A lariat is formed due to the GU at the 5’ end of the intron forming a phosphodiester bond with the branchpoint A The free 3’OH on exon 1 attacks the phosphodiester bond between the intron and exon 2 The exons are then linked

Signal at the Branch Along with consensus sequences at 5’- and 3’-ends of nuclear introns, branchpoint consensus sequences also occur Yeast sequence invariant: UACUAAC Higher eukaryote consensus sequence is more variable Branched nucleotide is final A in the sequence

Spliceosomes Splicing takes place on a particle called a spliceosome Yeast spliceosomes and mammalian spliceosomes have sedimentation coefficients of 40S and 60S Spliceosomes contain the pre-mRNA Along with snRNPs and protein splicing factors These recognize key splicing signals and orchestrate the splicing process

snRNPs Small nuclear RNAs coupled to proteins are abbreviated as snRNPs, small nuclear ribonuclear proteins The snRNAs (small nuclear RNAs) can be resolved on a gel: U1, U2, U4, U5, U6 All 5 snRNAs join the spliceosome to play crucial roles in splicing

U1 snRNP U1 snRNA sequence is complementary to both 5’- and 3’-splice site consensus sequences U1 snRNA base-pairs with these splice sites Brings the sites together for splicing is too simple an explanation Splicing involves a branch within the intron

Wild-Type and Mutant U1 snRNA Genetic experiments have shown that base pairing between U1 snRNA and 5’-splice site of mRNA precursor is necessary but not sufficient for binding

U6 snRNP U6 snRNP associates with the 5’-end of the intron by base pairing through the U6 RNA Occurs first prior to formation of lariat intermediate but after first step in splicing The association between U6 and splicing substrate is essential for the splicing process U6 also associates with U2 during splicing

A Model for interaction between a yeast 5’ splice site and U6 snRNA

U2 snRNP U2 snRNA base-pairs with the conserved sequence at the splicing branchpoint This base pairing is essential for splicing U2 also forms base pairs with U6 This region is called helix I Helps orient snRNPs for splicing 5’-end of U2 interacts with 3’-end of U6 This interaction forms a region called helix II This region is important in splicing in mammalian cells, not in yeast cells

Yeast U2 Base Pairing with Yeast Branchpoint Sequence

U5 snRNP U5 snRNA associates with the last nucleotide in one exon and the first nucleotide of the next exon This should result in the two exons lining up for splicing

U4 snRNP U4 base-pairs with U6 Its role seems to be to bind U6 When U6 is needed in a splicing reaction U4 is removed

snRNP Involvement in mRNA Splicing Spliceosomal complex contains: Substrate, U2, U5, and U6 The complex ready for the 2nd step in splicing can be drawn as a group II intron at same stage of splicing Spliceosomal snRNPs substitute for elements at center of catalytic activity of group II introns at same stage of splicing

Spliceosome Catalytic Activity Catalytic center of spliceosome appears to include Mg2+ and a base-paired complex of 3 RNAs: U2 snRNA U6 snRNA Branchpoint region of the intron Protein-free fragments of these RNAs can catalyze a reaction related to the first step in splicing

Spliceosome Assembly and Function Spliceosome is composed of many components – proteins and RNA These components assemble stepwise The spliceosome cycle includes sssembly, splicing activity, and disassembly By controlling assembly of the spliceosome, a cell can regulate quality and quantity of splicing and so regulate gene expression

Spliceosome Cycle Assembly begins with binding of U1 to splicing substrate forming a commitment complex, a unit committed to to splicing out the intron U2 joins the complex next, followed by the others U2 binding requires ATP U6 dissociates from U4 and displaces U1 at the 5’-splice site This step is ATP-dependent Activates the spliceosome Allows U1 and U4 to be released

snRNP Structure All snRNP’s have the same set of 7 Sm proteins Common targets of antibodies in patients with systemic autoimmune diseases Sm protein binds to a common Sm site on the snRNAs: AAUUUGUGG U1 snRNP has 3 specific proteins 70K has an Mr of 52 kD A has an Mr of 31 kD C has an Mr of 17.5 kD Sm proteins form a doughnut-shaped structure with a hole through the middle, like a flattened funnel

Sm Site and RNA Five snRNPs participate in splicing All contain a common set of 7 Sm proteins and several other proteins that are specific to snRNP Structure of U1 snRNP reveals that the Sm proteins form a doughnut-shaped structure to which the other proteins are attached

A Minor Spliceosome A minor class of introns with variant but highly conserved 5’-splice sites and branchpoints can be spliced with the help of a variant class of snRNAs Cells can contain minor snRNAs: U11 performs like U1 U12 acts like U2 U4atac and U6atac perform like U4 and U6 respectively

Commitment, Splice Site Selection and Alternative Splicing snRNPs do not have enough specificity and affinity to bind exclusively and tightly at exon-intron boundaries Additional splicing factors are needed to help snRNPs bind Some splicing factors are needed to bridge across introns and exons and so define these RNA elements

Exon and Intron Definition The spliceosome can recognize either exons or introns in the splicing commitment process, presumably by assembling splicing factors to bridge across exons or introns If exons are recognized it is exon definition If introns are recognized it is intron definition Splicing in a given organism typically uses either exon definition or intron definition

Commitment Commitment to splice at a given site is determined by an RNA-binding protein This protein binds to splicing substrate and recruits other spliceosomal components The first component to follow is U1 SR proteins SC35 and SF2/ASF commit splicing on human b-globin pre-mRNA and HIV tat pre-mRNA Part of the commitment involves attraction of U1 in some cases

Bridging Proteins and Commitment Yeast commitment complex has a branchpoint bridging protein (BBP) binds to: U1 snRNP protein at the 5’-end of the intron Mud2p near the 3’-end of the intron RNA near the 3’-end of the intron Bridges the intron and could play a role defining intron prior to splicing Mammalian BBP is SF1, may serve same bridging function

3’-Splice Site Selection Splicing factor Slu7 is required for correct 3’-splicing site selection Without Slu7, splicing to correct 3’-splice site AG is suppressed and splicing to aberrant AG’s within 30 nt of the branchpoint is activated U2AF is also required for 3’-splice site recognition 65-kD U2AF subunit binds to polypyrimidine tract upstream of 3’-splice site and 35-kD subunit binds to the 3’-splice site AG

Role of the RNA Polymerase II CTD C-terminal domain of the Rpb1 subunit of RNA polymerase II stimulates splicing of substrates that use exon definition This does not apply to those that use intron definition to prepare for splicing CTD binds to splicing factors and could assemble the factors at the end of exons to set them off for splicing

Alternative Splicing Transcripts of many eukaryotic genes are subject to alternative splicing This splicing can have profound effects on the protein products of a gene Can make a difference between: Secreted or membrane-bound protein Activity and inactivity Products of 3 genes in sex determination pathway of the fruit fly are subject to alternative splicing

Alternative Splicing in Drosophila sex determination

Sex-Specific Splicing Female-specific splicing of tra transcript gives: An active product that causes female-specific splicing of dsx pre-mRNA This produces a female fruit fly Male-specific splicing of tra transcript gives: An inactive product that allows male-specific splicing of dsx pre-mRNA This produces a male fruit fly

Tra and Tra-2 Tra and its partner Tra-2 act in conjunction with one or more other SR proteins to commit splicing at the female-specific splice site on the dsx pre-mRNA Commitment is probably the basis of most, if not all, alternative splicing schemes

Alternative Splicing Patterns Alternative splicing of the same pre-mRNA gives rise to very different products Alternative splicing patterns occur in over half of human genes Many genes have more than 2 splicing patterns, some have thousands

Control of Splicing Begin transcripts at alternative promoters Some exons can simply be ignored resulting in deletion of the exon Alternative 5’-splice sites can lead to inclusion or deletion of part of an exon Alternative 3’-splice sites can lead to inclusion or deletion of part of an exon A retained intron can be retained in the mRNA if it is not recognized as an intron Polyadenylation causes cleavage of pre-mRNA and loss of downstream exons

Silencing of Splicing What stimulates recognition of signals under only some circumstances? Exons can contain sequences – Exonic splicing enhancers (ESEs) stimulate splicing Exonic splicing silencers (ESSs) inhibit splicing

Reporter Construct Detects ESS Activity

Alternative Splicing Summary Alternative splicing is very common in higher eukaryotes It represents a way to get more than one protein product out of the same gene and a way to control gene expression in cells Such control is exerted by splicing factors that bind to splice sites and a branchpoint, and also by proteins that interact with ESEs, ESSs and intronic splicing elements

14.3 Self-Splicing RNAs Some RNAs could splice themselves without aid from a spliceosome or any other protein Tetrahymena 26S rRNA gene has an intron, splices itself in vitro Group I introns are a group of self-splicing RNAs Another group, Group II introns also have some self-splicing members

Group I Introns Group I introns can be removed in vitro with no help from protein Reaction begins with attack by a guanine nucleotide on the 5’-splice site Adds G to the 5’-end of the intron Releases the first exon Second step, first exon attacks the 3’-splice site Ligates 2 exons together Releases the linear intron Intron cyclizes twice, losing nucleotides each time, then linearizes a last time

Group I Intron: Tetrahymena 26S rRNA precursor

Group II Introns RNAs containing group II introns self-splice by a pathway using an A-branched lariat intermediate, like spliceosome lariats Secondary structures of the splicing complexes involving spliceosomal systems and group II introns are very similar