Chapter 14: RNA Splicing

simplified mechanism of splicing

1. 2'-OH group of A within intron attacks the phosphodiester bond linking the first extron to the intron 2. lariat is formed due to the GU at the 5' end of the intron, forming a phosphodiester bond with branch point A 3. free 3'OH on exon 1 attacks phosphodiester bond between the intron and exon 2 4. exons are then linked

components of canonical splicing pathway

1. E complex 2. A complex 3. B complex 4. C complex

mutually exclusive

1. Events that cannot happen at the same time 2. occurs in alternative splicing

mechanisms of silencer actions

1. HnRNPA1 binds to ESS within the exon and spreads through cooperative bonding until it occludes the ESE 2. PTB binds to the sequence flanking an exon and interacts with U1 at 5' splice site, which blocks U! to interact with the 3' splice site components

exon definition process

1. SR protein and ESE 2. SR proteins bound to the ESE sites recruit the splicing machinery to the nearby splicing site 3. SR recruits the U2AFs the the 3' splice site and U1 snRNP to the 5' site 4. mechanism that enhances the accuracy of splice-site selection

splicing activator

1. SR protein with arginine and serine rich RS domain and RNA-recognition motif 2. RS domains mediate the interactions between SR protein and the proteins within the splicing machinery

E complex in canonical splicing pathway

1. U1 snRNA recognizes 5' splicing site 2. U2AF made of 2 subunits that help protein bind to branch site

A complex in canonical splicing pathway

1. U2 snRNP binds to branch site and displaces BBP 2. A residue is not paired and available to react with 5' splicing site

U4 snRNP

1. U4 base pairs with U6 2. When U6 is needed in a splicing reaction, U4 is removed

coding sequence

1. a series of three nucleotide codons that specifies the linear sequence of amino acids in a polypeptide product 2. contiguous in bacteria and phage genes, but mosaic in eukaryotic genes

pluripotency

1. ability to differentiate into any cellular tissue other than the placenta 2. determined by an alternative splicing switch

components of guide RNA

1. anchor region: directs gRNA to the region of mRNA it will edit 2. editing region: determines the U insertion pattern 3. Poly-U stretch: probably tethers guide RNA to the purine rich sequences in the mRNA upstream of the edited region

RNA editing

1. another way of altering the sequence of an mRNA 2. type is deamination

U6 snRNP

1. associates with 5' end of intron by base pairing with u6 RNA 2. association occurs first prior to formation of lariat intermediate, but persists after first step in splicing 3. association between U6 and splicing substrates is essential for splicing process 4. U6 also associated with U2 during splicing

U5 snRNP

1. associates with last nucleotide in one exon and the first nucleotide of the next exon 2. should result in two exons lining up for splicing

U2 snRNP

1. base pairs with conserved sequence at the splicing branch point, which is essential for splicing 2. also forms base pairs with U2 that helps orient snRNPs for splicing 3. 5' end of U2 interacts with 3' end of U6

evidence for reshuffling exons

1. border between exons and introns with given gene often coincide with borders between domains 2. many genes have arisen from evolution in part via exon duplication and divergence 3. related exons are sometimes found in otherwise unrelated genes

B complex in canonical splicing pathway

1. brings together all three splicing sites 2. U4 and U6 snRNPS, along with U5 snRNP are join the complex, forming a tri-snRNP complex

solutions that enhance the accuracy of splice-site selection

1. co-transcription splicing 2. exon definition process

Group I introns

1. conserved secondary structure that includes a binding pocket that will accommodate any guanine nucleotide/nucleoside 2. contains internal guide sequence that base pairs with 5-splicing site sequence and thereby determines the precise site at which nucleophilic attack by G nucleotide takes place 3. can be converted to ribozymes

alternative splicing

1. different selection of exons can be generated from a given pre-mRNA 2. called isoforms 3. variant of splicing where exons can be deliberately skipped

branchpoint site

1. entirely within the intron and usually close to the 3' end 2. sequence necessary for splicing

errors in splice site recognition

1. exon skipping 2. pseudo splice-site recognition

advantage of intron

1. generate multiple protein products from a single gene 2. allows exons to be divided into several axons, therefore generated the possibility of reshuffling exons

what prevents splicing reaction from reversing itself

1. high concentration of G nucleotides that strongly favor forward reaction 2. intron can undergo further reaction that prevents it from participating in back reaction 3. 5' end of intron can bind along internal guide sequence, leading to third transesterification reaction 4. new bond formed with terminal G can hydrolyze spontaneously. intron is relinearized, but truncated and prevented from back-splicing reaction

splicing silencer

1. hnRPs (heterogenous nuclear ribonucleoprotein 2. include hnRNPA1, hnRNPI which bind to RNA, but lack Rs domain and can't recruit splicing machinery

how does information not find its way into mature RNA products of genes

1. introns are never transcribed: polymerase somehow jumps from one exon to another 2. introns are transcribed: the overlarge gene product will be cut down by removing introns

events in second stage of RNA splicing

1. mRNA maturation 2. removal of introns in a process called splicing

canonical splicing pathway

1. made of four different complexes 2. active site is formed primarily of RNA 3. snRNPs can be recycled 4. reaction can go backward and forward, but directionality is ensured because spliceosome is rapidly disassembled after second reaction takes place

classes of RNA splicing

1. nuclear pre-mRNA 2. Group II introns 3. Group I introns

co-transcription splicing

1. occurs when RNA polIII carries the various proteins for RNA processing 2. when the 5' splice site is encountered in the newly synthesized RNA, the factors that recognize that site are transferred from the polymerase carboxyl-terminal tail onto the RNA 3. once in place, the 5' splice site components are poised to interact with those other factors that bind to the next 3' splice site to be synthesized 4. the correct 3' splice site can be recognized before any competing sites downstream have been transcribed

mRNA transport

1. only certain RNAs are selected for transport-must have correct collection of proteins bound to it 2. proteins that indicate exon-intron boundaries indicate mRNA has been spliced appropriately. once in cytoplasm some proteins are shed, while others are taken on in readiness for translation 3. export requires energy, which is supplied by hydrolysis of GTP by Ran GTPase

spliceosome

1. particle where splicing takes place 2. contain the pre-mRNA 2. also contain snRNPs and protein splicing factors, which recognize key splicing signal and orchestrate the splicing process

RNA splicing

1. process of cutting introns out of immature RNAs and stitching together the exons to form the final product 2. precise-if not, could cause frameshift and result in the wrong protein

regulation of alternative splicing

1. regulated by activators and inhibitors 2. ESE or ESS: exonic splicing enhancer or repressor sites 3. ISI or ISS: intronic splicing enhancer or repressor sites

converting group I introns into ribozyme

1. relinearized intron retains active site. if it is provided with free Gs and a substrate that has sequence complementary to internal guide sequence, it will catalyze cleavage of substrate molecules 2. changing sequence of internal guide sequence will generate tailor-made ribonucleases

outcome of alternative splicing

1. secreted or membrane-bound protein 2. activity or inactivity

u1 snRNP

1. sequence is complementary to both 5' and 3'-splice site consensus sequences 2. splicing involves a branch within the intron 3. genetic experiments have shown that base pairing between u1 snRNA and 5' splice site of mRNA precursor is necessary, but not sufficient for binding

splicing signals

1. splicing must be precise 2. first 2 bases of introns are GU and last 2 are AG

mechanisms of mutually exclusive splicing

1. steric hindrance 2. combination of major and minor splice sites 3. nonsense-mediated decay

events in first stage of RNA splicing

1. synthesis of primary transcript product 2. this is an mRNA precursor containing introns copied from the gene if present 3. precursor is part of a pool of heterogenous nuclear RNAs (hnRNAs)

self-splicing intron

1. the intron itself folds into specific conformation within the precursor RNA and catalyzes the chemistry of its own release 2. occurs in the absence of any proteins or other RNA molecules

variants of splicing

1. trans-splicing 2. alternative splicing 3. minor spliceosome

mechanism of nuclear pre-mRNA splicing

1. two transesterification reactions at branch site A 2. same as pre-mRNA

trans-splicing

1. variant of splicing 2. two exons being joined reside on different RNA molecules

minor spliceosome

1. variant of splicing 2. use U11 and U12 instead of U1 and U2

minor (AT-AC) spliceosome

a minor class of introns with variant, but highly conserved 5'-splice sites and branch points that can be spliced with the help of a variant class of snRNAs

location of mRNA processing events

all events occur in nucleus before mRNA migrates to cytoplasm

intron

any region of the primary transcript that is not in a mature RNA

exon

any region of the primary transcripts that retains in a mature RNA

what determines sex of a fly

determined by alternative splicing events

guide RNA

directs the insertion and deletion of uridines

exon function

encode protein domains

intron late model

intron never exists in bacteria, but rather arose later in evolution

drosophila Dscam Gene

mutually exclusive splicing on a grand scale

purpose of exon shuffling

produces new proteins during evolution

Group II/I introns mechanism

two transesterification reactions at branch site G

intron early model

the intron existed in all organisms, but were lost from bacteria

lariat

The lasso-shaped structure formed during the removal of introns in mRNA processing

C complex in canonical splicing pathway

rearrangment occurs and U4 released from complex, allowing U6 to interact with U2 through RNA: RNA base pairing

snRNP

small nuclear RNAs coupled to proteins and abbreviated as small nuclear ribonuclear proteins