The mechanism of splicing of nuclear mRNA precursors

Chapter 14

Evidence for Split Genes

• Most higher eukaryotic genes coding for mRNA and tRNA are interrupted by unrelated regions called introns

• Exons are present surrounding the introns• Exons contain the sequences that finally

appear in the mature RNA product– Genes for mRNAs have been found with anywhere

from 0 to 362 exons– tRNA genes have either 0 or 1 exon

How do introns not find its way into mature RNA products of the genes? - RNA Splicing

• Introns are never transcribed– Polymerase somehow

jumps from one exon to another

• Introns are transcribed– Primary transcript result-

an overlarge gene product is cut down by removing introns

– This is correct process

RNA splicing

• Process of cutting introns out of immature RNAs and stitching together the exons to form final product is RNA splicing

• 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 large nuclear RNAs – hnRNAs

• Second stage:– mRNA maturation– Removal of introns in a process called splicing

Splicing Signals

• Splicing signals in nuclear mRNA precursors are remarkably uniform (exon/GU-intron-AG/exon)– 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 (Look at mammalian and yeast consensus sequences on page 403)

• Abnormal splicing can occur when the consensus sequences are mutated

Mechanism of Splicing of Nuclear mRNA Precursors

• Intermediate in nuclear mRNA precursor splicing is branched – looks like a lariat

• 2-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

Spliceosomes

• Splicing takes place on a particle called a spliceosome

• Yeast 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 5’- splice site consensus sequences

– U1 snRNA base-pairs with these splice sites

• Splicing involves a branch within the intron

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

• U6 also associates with U2 during splicing

U2 snRNP

• U2 snRNA base-pairs with the conserved sequence at the splicing branchpoint

• 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

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

snRNP Involvement in mRNA Splicing

• Spliceosomal complex contains:– Substrate – U2– U5– U6– All snRNP are made up of

same seven set of proteins called Sm proteins

Spliceosome Assembly and Function

• Spliceosome is composed of many components – proteins and RNA

• These components assemble stepwise• The spliceosome cycle:

– Assembly

– Function

– 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 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

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

Yeast Two-Hybrid Assay

Intron-Bridging Protein-Protein Interactions

• Branchpoint bridging protein binds to U1 snRNP protein

• Comparison of yeast to mammalian complexes is seen at right

Role of the RNA Polymerase II CTD

• CTD binds to splicing factors and could assemble the factors at the end of exons to set them off for splicing (figure 14.37)

• Questions 27, 28 and 31 - Homework

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

Alternative Splicing Patterns-Pg 432

• 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

What stimulates recognition of signals under only some circumstances? - Silencing of Splicing

• Exons can contain sequences – – Exonic splicing

enhancers (ESEs) stimulate splicing

– Exonic splicing silencers (ESSs) inhibit splicing

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– 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

Linear Introns

• 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 II Introns

• RNAs containing group II introns self-splice by a pathway using an A-branched lariat intermediate - like spliceosome lariats

Types of Alternative 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

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