RNA splicingRNA splicingBy xiao yi By xiao yi

生物学基地班生物学基地班200431060010200431060010

In most cases of eukaryotic gene, In most cases of eukaryotic gene, the coding sequences is interruptthe coding sequences is interrupted by noncoding sequencesed by noncoding sequences• The coding sequences are called exonsThe coding sequences are called exons• The noncoding sequences are called intrThe noncoding sequences are called intr

onsons

Before translation, the introns of Before translation, the introns of pre-RNA must be removed, and thpre-RNA must be removed, and this process is called RNA splicingis process is called RNA splicing

• RNA splicing: the process by which intRNA splicing: the process by which introns are removed from the pre-mRNA.rons are removed from the pre-mRNA.

• Alternative splicing : some pre-mRNAs Alternative splicing : some pre-mRNAs can be spliced in more than one way , can be spliced in more than one way , generating alternative mRNAsgenerating alternative mRNAs. . 60% of 60% of the human genes are spliced in this mthe human genes are spliced in this manner.anner.

The chemistry of RNA The chemistry of RNA splicingsplicing

• Sequences within the RNA Determine WSequences within the RNA Determine Where Splicing Occurshere Splicing Occurs

• The borders between introns and exons The borders between introns and exons are marked by specific nucleotide sequeare marked by specific nucleotide sequences within the pre-mRNAs.nces within the pre-mRNAs.

the exon-intron boundary at the 5’ end of the intron is called 5’ splicing site

the exon-intron boundary at the 3’ end of the intron is called 3’ splicing site

Branch point site

an A close to the 3’ end of the intron, which is followed by a polypyrimidine tract (Py tract).

The intron is removed in a Form CalThe intron is removed in a Form Called a Lariat as the Flanking Exons aled a Lariat as the Flanking Exons are joinedre joined

• RNA splicing is achieved by two successiRNA splicing is achieved by two successive transesterification reactions ve transesterification reactions

Step 1Step 1

The OH of the conserved A at the brancThe OH of the conserved A at the branch site attacks the phosphoryl group of h site attacks the phosphoryl group of the conserved G in the 5’ splice site. the conserved G in the 5’ splice site. As a result, the 5’ exon is released anAs a result, the 5’ exon is released and the 5’-end of the intron forms a thrd the 5’-end of the intron forms a three-way junctionee-way junction structure. structure.

Step 2Step 2

The OH of the 5’ exon attacks the phosThe OH of the 5’ exon attacks the phosphoryl group at the 3’ splice site. As phoryl group at the 3’ splice site. As a consequence, the 5’ and 3’ exons a consequence, the 5’ and 3’ exons are joined and the intron is liberated iare joined and the intron is liberated in the shape of a lariat.n the shape of a lariat.

Exons from different RNA moleculeExons from different RNA molecules can be fused by Trans-splicings can be fused by Trans-splicing

• The only difference is that the other The only difference is that the other product---the lariat in the standard product---the lariat in the standard reaction---is, in trans splicing, is a Y reaction---is, in trans splicing, is a Y shaped branch structure insteadshaped branch structure instead

THE SPLICESOME THE SPLICESOME MACHINERYMACHINERY

• RNA splicing is carried out by a large cRNA splicing is carried out by a large complex called spliceosomeomplex called spliceosome

• The spliceosome comprises about 150 The spliceosome comprises about 150 proteins and 5 proteins and 5 snRNAs snRNAs

• Many functions of the spliceosome are Many functions of the spliceosome are carried out by its RNA components.carried out by its RNA components.

The five RNAsThe five RNAs

• U1, U2, U4, U5, and U6 are called smalU1, U2, U4, U5, and U6 are called small nuclear RNAs (snRNAs)l nuclear RNAs (snRNAs)

• The complexes of snRNA and proteins The complexes of snRNA and proteins are called small nuclear ribonuclear pare called small nuclear ribonuclear proteins (snRNP)roteins (snRNP)

Three roles of snRNPs in splicinThree roles of snRNPs in splicingg• They recognize the 5’ splicing site They recognize the 5’ splicing site

and the branch siteand the branch site

• They bring those site together as They bring those site together as requiredrequired

• They catalyze (or help catalyze) the They catalyze (or help catalyze) the RNA cleavage and joining reactionsRNA cleavage and joining reactions

SPLICING SPLICING PATHWAYSPATHWAYS•AssemblyAssembly

•RearrangementRearrangement

•catalysiscatalysis

Assembly step 1Assembly step 1

1. 1. U1U1 recognize 5’ splice site. recognize 5’ splice site. 2. One subunit of 2. One subunit of U2AFU2AF binds to binds to Py tractPy tract

and the other to the and the other to the 3’ splice site3’ splice site. Th. The former subunits interacts with BBP ae former subunits interacts with BBP and helps it bind to the branch point.nd helps it bind to the branch point.

3. 3. Early (E) complex isEarly (E) complex is formed formed

Assembly step 2Assembly step 2

1. 1. U2 binds to the branch site, and then U2 binds to the branch site, and then A complex is formed.A complex is formed.

2. The base-pairing between the U2 and 2. The base-pairing between the U2 and the branch site is such that the branch the branch site is such that the branch site A is extruded. This A residue is avasite A is extruded. This A residue is available to react with the 5’ splice site.ilable to react with the 5’ splice site.

Assembly step 3Assembly step 3

1.1. U4, U5 and U6 form the tri-snRNP ParU4, U5 and U6 form the tri-snRNP Particle. ticle.

2. With the 2. With the entry of the tri-snRNP, the A entry of the tri-snRNP, the A complex is converted into the B complcomplex is converted into the B complex.ex.

Assembly step 4Assembly step 4

U1 leaves the complex, and U6 replaces U1 leaves the complex, and U6 replaces it at the 5’ splice site.it at the 5’ splice site.

U4 is U4 is released from the complex, allowireleased from the complex, allowing U6 to interact with U2 .This arrangng U6 to interact with U2 .This arrangement called the C complex.ement called the C complex.

U1 leaves the complex, and U6 replaces it at the 5’ splice site.U4 is released from the complex, allowing U6 to interact with U2 .This arrangement called the C complex.

Catalysis Step 1Catalysis Step 1

Formation of the C complex produces the actiFormation of the C complex produces the active site, with U2 and U6 RNAs being brought ve site, with U2 and U6 RNAs being brought togethertogether

Formation of the active site juxtaposes the Formation of the active site juxtaposes the 5’ splice site of the pre-mRNA and the bran5’ splice site of the pre-mRNA and the branch site, allowing the branched A residue to ach site, allowing the branched A residue to attack the 5’ splice site to accomplish the fittack the 5’ splice site to accomplish the first transesterfication reaction.rst transesterfication reaction.

Catalysis Step 2:

U5 snRNP helps to bring the two exons together, and aids the second transesterification reaction, in which the 3’-OH of the 5’ exon attacks the 3’ splice site.

Self-splicing introns reveal that RNSelf-splicing introns reveal that RNA can catalyze RNA splicingA can catalyze RNA splicing

TABLE 13-1TABLE 13-1 Three class of RNA Three class of RNA SplicingSplicing

ClassClass AbundanceAbundance MechanismMechanism Catalytic Catalytic MachinerMachiner

yy

NucleaNuclear pre-r pre-mRNAmRNA

Very common; used Very common; used for most eukaryotic for most eukaryotic

genesgenes

Two Two transesterifictransesterific

ation ation reactions; reactions;

branch site Abranch site A

Major Major spliceosospliceoso

meme

Group II Group II intronsintrons

Rare; some eu-Karyotic Rare; some eu-Karyotic genes from organelles genes from organelles

and prokaryotesand prokaryotes

Same as pre-Same as pre-mRNAmRNA

RNA enzymRNA enzyme encoded e encoded

by intron (rby intron (ribozyme)ibozyme)

Group I iGroup I intronsntrons

Rare; nuclear rRNA in sRare; nuclear rRNA in some eukaryotics, orgaome eukaryotics, organlle genes, and a few pnlle genes, and a few p

rokaryotic genesrokaryotic genes

Two transesteriTwo transesterific-ation reactiofic-ation reactions; exogenous ns; exogenous

GG

Same as grSame as group II introoup II intro

nsns

• When we examine the group 1 and grouWhen we examine the group 1 and group 2 self-splicing, we find the intron itself p 2 self-splicing, we find the intron itself folds into a specific conformation within folds into a specific conformation within the precursor RNA and catalyze the chethe precursor RNA and catalyze the chemistry of its own releasemistry of its own release

• The chemistry of group II intron spliciThe chemistry of group II intron splicing and RNA intermediates produced ang and RNA intermediates produced are the same as that of the nuclear pre-re the same as that of the nuclear pre-mRNA.mRNA.

Group I introns release a linear intrGroup I introns release a linear intron rather than a lariaton rather than a lariat

• Instead of a branch point A residue, they Instead of a branch point A residue, they use a free G nucleotide or nucleoside. Thuse a free G nucleotide or nucleoside. This G species is bound by the RNA and its is G species is bound by the RNA and its 3’ OH group is presented to the 5’ spli3’ OH group is presented to the 5’ splicing site. Here fuses the G to the 5’ end cing site. Here fuses the G to the 5’ end of the intron. The freed 3’ end attacks tof the intron. The freed 3’ end attacks the 3’ splicing site. In this case the introhe 3’ splicing site. In this case the intron is linear rather than a lariat structuren is linear rather than a lariat structure

Two kinds of splice-site recognition errors

•Splice sites can be skipped.•“Pseudo” splice sites could be mista

kenly recognized, particularly the 3’ splice site.

Two ways to enhance the Two ways to enhance the accuracyaccuracy

• RNA polymerase carries with it various pRNA polymerase carries with it various proteins with roles in RNA processingroteins with roles in RNA processing

• SR (serine argenine anthentic) proteins SR (serine argenine anthentic) proteins bind to sequences to called exonic splicibind to sequences to called exonic splicing enhancers (ESEs) within the exonsng enhancers (ESEs) within the exons

SR proteins are essential for splicing

• They ensure the accuracy and efficiency They ensure the accuracy and efficiency of constitutive splicing.of constitutive splicing.

• They also regulate alternative splicingThey also regulate alternative splicing

• They come in many varieties, some They come in many varieties, some controlled by physical signals, others controlled by physical signals, others constitutively active. Some are constitutively active. Some are expressed preferentially in certain cell expressed preferentially in certain cell types and control splicing in cell-type types and control splicing in cell-type specific patterns.specific patterns.

Alternative splicingAlternative splicing

• RNAs can be spliced in alternative ways tRNAs can be spliced in alternative ways to generate different mRNAs and, thus, dio generate different mRNAs and, thus, different protein productsfferent protein products

There are five ways to splice a There are five ways to splice a RNARNA

Alternative splicing can be either constitutive or regulated•Constitutive alternative splicing: mor

e than one product is always made from a pre-mRNA

•Regulative alternative splicing: different forms of mRNA are produced at different time, under different conditions, or in different cell or tissue types

Alternative splicing is Alternative splicing is regulated by activators and regulated by activators and repressorsrepressors• Proteins that regulate splicing bins to spProteins that regulate splicing bins to sp

ecific sites called exonic (or intronic) spliecific sites called exonic (or intronic) splicing enhancers (ESE or ISE) or silencers cing enhancers (ESE or ISE) or silencers (ESS and ISS)(ESS and ISS)

• The former enhance and the latter repreThe former enhance and the latter repress, splicing at nearby splice sitesss, splicing at nearby splice sites

• The SR proteins bind RNA using one doThe SR proteins bind RNA using one domain----RNA-recognition motif (RRM)main----RNA-recognition motif (RRM)

• Each SR protein use RS domain which is Each SR protein use RS domain which is rich in arginine and serine to mediate intrich in arginine and serine to mediate interactions between the SR protein and preractions between the SR protein and proteins within the splicing machineryoteins within the splicing machinery

• Heterogeneous nuclear ribonucleoproteHeterogeneous nuclear ribonucleoprotein in (hnRNP) bind to RNA but lack the RS do(hnRNP) bind to RNA but lack the RS domain so cannot recruit splicing machinamain so cannot recruit splicing machinary, instead, they repress the use of those ry, instead, they repress the use of those sitessites

hnRNPI protein repress splicing bhnRNPI protein repress splicing by two ways:y two ways:• hnRNPI bind to each end of exon, then ihnRNPI bind to each end of exon, then i

nteract with each other, looping out the nteract with each other, looping out the exonexon

• hnRNPI coat the RNA across the whole ehnRNPI coat the RNA across the whole exon, making the exon invisible to the splixon, making the exon invisible to the splicing machinarycing machinary

A small group of intron are splicA small group of intron are spliced by minor spliceosomeed by minor spliceosome• This spliceosome works on a minority This spliceosome works on a minority

of exons, and those have distinct splicof exons, and those have distinct splice-site sequence. e-site sequence.

• The chemical pathway is the same as tThe chemical pathway is the same as the major spliceosome.he major spliceosome.

Exon shufflingExon shuffling

Exons are shuffled by recombination to produce gene encoding new proteins

1.1. Introns early modelIntrons early model – introns existed i – introns existed in all organisms but have been lost fron all organisms but have been lost from bacteria.m bacteria.

2.2. Intron late modelIntron late model – introns never exis– introns never existed in bacteria but rather arose later ited in bacteria but rather arose later in evolution.n evolution.

Why have the intronsWhy have the introns been retaibeen retained in eukaryotes?ned in eukaryotes?

1. The need to remove introns, allows fo1. The need to remove introns, allows for alternative splicing which can generr alternative splicing which can generate multiple proteins from a single geate multiple proteins from a single gene. ne.

2. Having the coding sequence of genes 2. Having the coding sequence of genes divided into several exons allows new divided into several exons allows new genes to be created bygenes to be created by reshuffling exoreshuffling exonn..

Three observations suggest exoThree observations suggest exon shuffling actually occurn shuffling actually occur1. The borders between exons and intro1. The borders between exons and intro

ns within a gene often coincide with tns within a gene often coincide with the boundaries between domains withihe boundaries between domains within the protein encoded by that gene.n the protein encoded by that gene.

2. Many genes, and proteins they encod2. Many genes, and proteins they encode, have apparently arisen during evolue, have apparently arisen during evolution in part via exon duplication and dtion in part via exon duplication and divergence.ivergence.

• 3. Related exons are sometimes found 3. Related exons are sometimes found in unrelated genes.in unrelated genes.

RNARNA EDITINGEDITING

• RNA editing is another way of RNA editing is another way of changing the sequence of an mRNAchanging the sequence of an mRNA

1. Site specific deamination1. Site specific deamination ::

1. A specifically targeted1. A specifically targeted C C residue withiresidue within mRNA is converted inton mRNA is converted into UU by the deaby the deaminaseminase..

2. The process occurs only in certain tiss2. The process occurs only in certain tissues or cell types and in a regulated maues or cell types and in a regulated manner.nner.

3. 3. Adenosine deaminationAdenosine deamination also occurs in cells. also occurs in cells. The enzymeThe enzyme ADAR ADAR (adenosine deaminase ac(adenosine deaminase acting on RNA) convert A into Inosine. Insone ting on RNA) convert A into Inosine. Insone can base-pair with C, and this change can alcan base-pair with C, and this change can alter the sequence of the protein. ter the sequence of the protein.

4. An ion channel expressed in mammalian br4. An ion channel expressed in mammalian brains is the target of Adenosine deamination.ains is the target of Adenosine deamination.

2 Guide RNA-directed uridine in2 Guide RNA-directed uridine insertion or deletion.sertion or deletion.

1. This form of RNA editing is found in th1. This form of RNA editing is found in the mitochondria of trypanosomes.e mitochondria of trypanosomes.

2. Multiple Us are inserted into specific r2. Multiple Us are inserted into specific region of mRNAs after transcription (or egion of mRNAs after transcription (or US may be deleted).US may be deleted).

3. The addition of Us to the message cha3. The addition of Us to the message changes codons and reading frames, comnges codons and reading frames, completely altering the “meaning” of thpletely altering the “meaning” of the message.e message.

4. Us are inserted into the4. Us are inserted into the message bymessage by gguide RNAsuide RNAs ((gRNAsgRNAs) .) .

Guide RNAsGuide RNAs

• gRNAs range from 40 to 80 gRNAs range from 40 to 80 nucleotides in length and are nucleotides in length and are encoded by genes distinct from those encoded by genes distinct from those that encode the mRNAs they act onthat encode the mRNAs they act on

• gRNAs have three regions:gRNAs have three regions:

1.Anchor1.Anchor

2.Editing region2.Editing region

3.poly-U stretch3.poly-U stretch

mRNA transportmRNA transport

•Once processed, mRNA is Once processed, mRNA is packaged and exported from the packaged and exported from the nucleus into the cytoplasm for nucleus into the cytoplasm for translationtranslation

•Movement from the nucleus to the cytoplasm is an active and carefully regulated process.

•The damaged, misprocessed and liberated introns are retained in the nucleus and degraded.

• A typical mature mRNA carries a collection of proteins that identifies it as being ready for transport.

• Export takes place through the nuclear pore complex.

Once in the cytoplasm, some proteins are discarded and are then imported back to the nucleus for another cycle of mRNA transport. Some proteins stay on the mRNA to facilitate translation.

Thank youThank you

By xiao yiBy xiao yi200431060010200431060010