Copyright The McGraw-Hill Companies.Inc.Permission required for reproduction or display. Chapter 14 Messenger RNA Processing I:Splicing Dr.David Spector/Peter Arnold,Inc
Chapter 14 Messenger RNA Processing I: Splicing
14.1 Genes in Pieces Consider the sequence of the human B-globin (β-球蛋白)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 Called intervening sequences(插入序列，IVSs) - Usually called introns(内含子) Parts of the gene making sense -Coding regions - Eons(外显子) Some lower eukaryotic genes have no introns 14-2
14-2 14.1 Genes in Pieces ✓ Consider the sequence of the human b-globin (b-球蛋白) 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 – Called intervening sequences (插入序列, IVSs) – Usually called introns (内含子) ✓ Parts of the gene making sense – Coding regions – Exons (外显子) ✓ Some lower eukaryotic genes have no introns
RNA Splicing Introns are present in genes but not in mature RNA Process of cutting introns out of immature RNAs and stitching together the exons to form final product is RNA splicing (RNA 接) 14-3
14-3 RNA Splicing ✓Introns are present in genes but not in mature RNA ✓Process of cutting introns out of immature RNAs and stitching together the exons to form final product is RNA splicing (RNA剪 接)
Splicing Outline CopyrightTe MeGraw-Hll Co Start of transcription √Introns are Intron 1 Intron 2 transcribed along Gene: Exon 1 Exon 2 Exon 3 with exons in the Transcription primary transcript Intron 1 Intron 2 √Introns are removed Primary transcript: Exon 1 Exon 2 Exon 3 as the exons are Splicing spliced together Mature transcript: Exon 1 Exon 2 Exon 3 14-4
14-4 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 large nuclear RNAs- heterogeneous nuclear RNA(核不均一RNA,hnRNAs) √Second stage: -mRNA maturation - Removal of introns in a process called splicing 14-5
14-5 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 – heterogeneous nuclear RNA (核不均一RNA, hnRNAs) ✓ Second stage: – mRNA maturation – Removal of introns in a process called splicing
Splicing Signals(剪接信号) Splicing signals in nuclear mRNA precursors are remarkably uniform -First 2 bases of introns are GU Last 2 are AG -Exon/GU-intron-AG/exon 5'-and 3'-splice sites have consensus sequences extending beyond GU and AG motifs 一 5'-AG/GUAAGU-intron-YNCURAC-YnNAG/exon Y:pyrimidine;Yn:about 9 pyrimidine;R:purine; Whole consensus sequences are important to proper splicing Abnormal splicing can occur when the consensus sequences are mutated 14-6
14-6 Splicing Signals (剪接信号) ✓ Splicing signals in nuclear mRNA precursors are remarkably uniform – First 2 bases of introns are GU – Last 2 are AG – Exon/ GU-intron- AG/ exon ✓ 5’- and 3’-splice sites have consensus sequences extending beyond GU and AG motifs – 5’- AG/ GUAAGU-intron- YNCURAC-YnNAG/ exon Y: pyrimidine; Yn: about 9 pyrimidine; R: purine; ✓ 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(套索) √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 14-7
14-7 14.2 Mechanism of Splicing of Nuclear mRNA Precursors ✓ Intermediate in nuclear mRNA precursor splicing is branched – looks like a lariat (套索) ✓ 2-step model – 2’-OH group of adenosine nucleotide in middle of intron attacks phosphodiester bond between 1 st 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 Excised intron has a 3'-OH group Phosphorus atom between Step 1 2 exons in spliced product comes from 3'-splice site AGD Intermediate and spliced intron contain a branched Step 2 nucleotide -AG-OH Branch involves 5'-end of intron binding to a site within the intron 14-8
14-8 Simplified Mechanism of Splicing ✓ Excised intron has a 3’-OH group ✓ Phosphorus atom between 2 exons in spliced product comes from 3’-splice site ✓ Intermediate and spliced intron contain a branched nucleotide ✓ Branch involves 5’-end of intron binding to a site within the intron
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 Wild-type Spliced Mutant #1 Not spliced Mutant#2 Aberrantly splicec Mutant #3 Aberrantly spliced 14-9
14-9 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(剪接体) Splicing takes place on a particle called a spliceosome Spliceosomes contain the pre-mRNA -Along with snRNPs (small nuclear ribonuclearprotein,核内小核糖核蛋白体)and protein splicing factors -These recognize key splicing signals and orchestrate(组织)the splicing process 14-10
14-10 Spliceosomes (剪接体) ✓Splicing takes place on a particle called a spliceosome ✓Spliceosomes contain the pre-mRNA – Along with snRNPs (small nuclear ribonuclearprotein, 核内小核糖核蛋白体) and protein splicing factors – These recognize key splicing signals and orchestrate (组织) the splicing process