Chapter 13 生物学基地班 孙钒 RNA Splicing

OUTLINE The Chemistry of RNA Splicing The Spliceosome Machinery Splicing Pathways (important) Alternative Splicing (important) Exon Shuffling RNA Editing mRNA Transport

Key Words Exon : coding sequences Intron : intervening sequences Pre-mRNA: the primary transcript of DNA RNA splicing : process that intron are moved from the pre-RNA Spliceosome : a huge molecular “machine” catalyse RNA splicing Alternative splicing :some pre-mRNAs can be spliced in more than one way,and produce alternative mRNAs.

Topic 1 The Chemistry of RNA Splicing Sequences within the RNA determine where splicing occurs The intron is moved in a form called Lariat as the flanking exons are joined Exons from different RNA molecules can be fused by trans-splicing

one: Sequences within the RNA determine where splicing occurs The borders between introns and exons are marked by specific nucleotide sequences ( GU—AG Law ) within the pre-mRNAs. Py-tract: rich in pyrimidine

Notice GU in 5` splicing site, AG in 3` splicing site and A in branch point site are the most conserved sequences, and they are all in the intron. These sequences are important for the distinguish between intron and exon,remove of intron,linkage of exons and delineate where splicing will occur.

Two :The intron is moved in a form called Lariat as the flanking exons are joined RNA splicing consists of two successive transesterification reaction.

Reaction 1 : The OH of the conserved A at the branch site attacks the phosphoryl group of the conserved G in the 5’ splice site. As a result, the 5’ exon is released and the 5’- end of the intron forms a three-way junction structure.

Reaction 2 : The OH of the 5’ exon attacks the phosphoryl group at the 3’ splice site. As a consequence, the 5’ and 3’ exons are joined and the intron is liberated in the shape of a lariat.

The structure of three-way junction In addition to the 5` and 3` backbone linkages,a third phosphodiester extends from the 2`OH of that A to create a three-way junction.

Reaction 2 Ensure the splicing only goes forward: One :an increase in entropy. Two :the excised intron lariat is rapidly degraded after its removal.

Notice In the two reactions, there is no net gain in the number of chemical bonds. So no energy is demanded by the process. But, we see a large amount of ATP is consumed during the splicing reaction. Why? This energy is required to properly assemble and operate the splicing machinery, not for the chemistry.

Three : Exons from different RNA molecules can be fused by trans-splicing Trans-splicing: the process in which two exons carried on different RNA molecules can be spliced together. This process is rare.But all mRNAs in the nematode worm undergo trans- splicing.

Topic 2 : The Spliceosome Machinery RNA splicing is carried out by a large complex called spliceosome Spliceosome is a complex that mediates splicing of introns from pre-mRNA. And it comprises about 150 proteins and 5 snRNAs. Many functions of the spliceosome are carried out by its RNA components.

The five RNAs (U1, U2, U4, U5, and U6, nt) are called small nuclear RNAs (snRNAs). The complexes of snRNA and proteins are called small nuclear ribonuclear proteins (snRNPs). The spliceosome is the largest snRNP, and the exact makeup differs at different stages of the splicing reaction. Different snRNPs come and go at different times,each carrying out particular functions in the reaction.

Three roles of snRNPs in splicing 1. Recognizing the 5’ splice site and the branch site. 2. Bringing those sites together. 3. Catalyzing (or helping to catalyze) the RNA cleavage. RNA-RNA, RNA-protein and protein-protein interactions are all important during splicing

RNA-RNA interactions between different snRNPs, and between snRNPs and pre-mRNA Branch-point binding protein

Topic 3 : Splicing Pathways Assembly, rearrangement, and catalysis within the spliceosome: the splicing pathway Self-splicing introns reveal that RNA can catalyze RNA splicing Group I introns release a linear intron rather than a lariat How does spliceosome find the splice sites reliably

One : Assembly, rearrangement, and catalysis within the spliceosome: the splicing pathway Steps of splicing pathway: Assembly : step one 1. U1 recognize 5’ splice site. 2. One subunit of U2AF binds to Py tract and the other to the 3’ splice site. The former subunits interacts with BBP and helps it bind to the branch point. 3. Early (E) complex is formed

Assembly Step two 1.With the help of U2AF, U2 binds to the branch site replacing of BBP, and then A complex is formed. 2. The base-pairing between the U2 and the branch site is such that the branch site A is extruded. This A residue is available to react with the 5’ splice site.

Assembly Step three 1. U4, U5 and U6 form the tri-snRNP Particle. 2. With the entry of the tri-snRNP, the A complex is converted into the B complex.

Assembly Step four 1, U1 leaves the complex, and U6 replaces it at the 5’ splice site. 2, U4 is released from the complex, allowing U6 to interact with U2.This arrangement called the C complex.

Catalysis Step one 1, Formation of the C complex produces the active site, with U2 and U6 RNAs being brought together

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

Catalysis Step two 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. Step three Release of the mRNA product and the snRNPs

Two : Self-splicing introns reveal that RNA can catalyze RNA splicing Self-splicing introns: the intron itself folds into a specific conformation within the pre-mRNA and catalyzes the chemistry of its own release (recall RNA enzyme ) and the exon ligation. Practical definition for self-splicing introns: the introns that can remove themselves from pre- RNAs in the test tube in the absence of any proteins or other RNAs. Two classes of self-splicing introns, group I and group II self-splicing introns.

Three classes of RNA splicing

Notice :The chemistry of group II intron splicing and RNA intermediates produced are the same as that of the nuclear pre- mRNA.

Three : Group I introns release a linear intron rather than a lariat Instead of using a branch point A, group I introns use a free G to attack the 5’ splice site. This G is attached to the 5’ end of the intron.The 3’-OH group of the 5’ exon attacks the 5’ splice site. The two-step transesterification reactions are the same as that of splicing of the group II intron and pre-mRNA introns.

Three classes of RNA splicing

Group 1 intron structure A complex secondary structure Share a conserved secondary structure, which includes an “internal guide sequence” base- pairing with the 5’ splice site sequence in the upstream exon.

Group 1 intron structure The tertiary structure contains a binding pocket that will accommodate the guanine nucleotide or nucleoside cofactor Bind any G-containing ribonucleotide.

Steps of group 1 intron splicing free guanosine binds in the guanine-binding pocket 3`OH of guanosine attacks phosphate at 5`end of intron 3`OH of exon 1 attack 5`phosphate of exon 2 Intron is released

Internal guide sequence base pairs with 5`end of intron 3`guanosine binds in guanine-binding pocket 3`OH of bound guanosine attacks phosphate to the right of IGS Bond between 3`G and 5` phosphate hydrolyzes, leaving inactive intron

The similarity of the structures of group II introns and U2-U6 snRNA complex formed to process first transesterification

Four : How does spliceosome find the splice sites reliably splice-site recognition is prone to 2 kinds of errors: ★ Splice sites can be skipped. ★ some site close in sequence but not legitimate splice site,could be mistakenly recognized. For example,“Pseudo” splice sites could be mistakenly recognized and pair with component at 5`site, particularly the 3’ splice site.

Error produced by mistakes in splice- site selection

Two ways to enhance the accuracy of the splice-site selection 1, the C-terminal tail of the RNA polymerase II carries various splicing proteins. when a 5`splice site is encountered in the newly synthesized RNA, those components are transferred from the Pol II C-terminal tail onto the RNA.

Once in place,the 5`splice site components are poised to interact with those that bind to the next 3`splice siteto be synthesized. Thus,the correct 3`splice site can be recognized before any competing sites further downstream have been transcribed. This co-transcriptional loading process greatly diminishes the likelihood of exon skipping.

2, a second mechanism guides against the use of incorrect sites by ensuring that splice sites close to exons are recognized preferentially. SR proteins bind to sequences called exonic splicing enhancers (ESEs) within the exons. SR bound to ESE interacts with components of the splicing machinery,recruiting them to the nearby splicing sites. In this way, the machinery binds more efficiently to the nearby sites than to incorrect sites not close to exons.

SR protein recruits spliceosome components to the 5`and 3` splice sites SR proteins bind to ESEs,recruit U2AF and U1snRNP to the downstream 5`and upstream 3`splice sites respectively. This initiates the assembly of the splice machinery on the correct sites and splice can proceed as outlined earlier.

SR proteins function Ensure the accuracy and efficiency of constitutive splicing. Regulate alternative splicing. They come in many varieties,controlled by physiological signals and constitutively active. And some express preferential in certain cell types and control splicing in cell-type specific patterns.

Topic 4 : Alternative splicing Single gene can produce multiple products by alternative splicing Alternative splicing is regulated by activators and repressors A small group of introns are spliced by an spliceosome composed of a different set of snRNPs

One : Single gene can produce multiple products by alternative splicing Alternative splicing many genes in higher eukaryotes encode RNAs that can be spliced in alternative ways to generate two or more different mRNAs and,different protein products.

As we know,exons are not skipped and splice sites not ignored, why does alternative splicing occur so often ? Answer : some splice sites are used only some of the time,leading to the production of different versions of the RNA from different transcripts of the same gene.

Five ways to splice an RNA

Alternative splicing can be either constitutive or regulated. Constitutive : more than one product from the same gene Regulated : different products are generated at different times, under different conditions, or in different cell or tissue type.

Constitutive alternative splicing Splicing of the monkey SV40 T antigen RNA. high level SF2/ASF

Two : Alternative splicing is regulated by activators and repressors Proteins that regulate splicing bind to specific sites called exonic (or intronic ) splicing enhancers or silencers. Protein + specific sequence can guide elements of spliceosome to exons.

SR protein family is large and diverse, has specific roles in regulated alternative splicing by directing the splicing machinery to different splice sites under different conditions. The presence or activity of a given SR can determine whether a particular splice site is used in particular cell type,or at a particular stage of development.

Regulated alternative splicing

RNA-recognition motif :RRM RS domain: rich in Arg and Binding RNA Ser. Found at C-terminal end of protein,mediates interactions between SR and the pro. within the splicing machinery, recruiting them to a nearby splice site. RS protein

An example of repressors: inhibition of splicing by hnRNPI Repressor are hnRNPs. They can bind RNA`s sliencer and repress the use of those sites. They don`t have RS domain,so can`t recruit spliceosome. By binding sites, this blocking can repress splice. Conceal exon as a loop Coat exon entirely to avoid splicing

The second way alternative splicing can be used as an on/off switch is by regulating the use of an intron, which, when retained in the mRNA,the mRNA will be never transported out of nucleous and be never translated.

Three : A small group of introns are spliced by an spliceosome composed of a different set of snRNPs Higher eukaryotes use the major splicing machinery :we have discussed before, and some pre-mRNAs are spliced by a low-aboundance form of spliceosome. The rare form contains some components common to major spliceosome but other unique. U11 and U12 have the same roles in the splicing reaction as U1 and U2, but they recognize distinct sequences. U4 and U6 share the same names but their snRNPs are distinct. U5 is identical.

The minor spliceosome recognizes rarely occurring introns having consensus sequences distinct from the sequences of most pre-mRNA. This intron contains 5` AT and 3`AC ( AT-AC low ), so the new form is known as the AT-ACspliceosome. Later,we discover that it also recognizes GT- AG.

The ability of the snRNAs and splice site sequences to base-pair is conserved, not any sequences within either. Although the different splice sites and branch site,the two forms of spliceosome share the same chemical pathway to remove introns.

Topic 5 Exon Shuffling Exons are shuffled by recombination to produce genes encoding new proteins. All eukaryotes have introns which rare in the bacteria. Two model: intron early model: all organism have introns, and bacteria lost them. intron late model: introns were inserted into genes by a transposon-like machanism

Why introns can exist in eukaryotes?? First :the presence of introns and the need to remove them,allow for alternative splicing which can generate multiple proteins from a gene. Second :having the coding sequence of genes divided into several exons allows new gene to be created by reshuffling exons.

Three observations 1. The borders between exons and introns within a gene often coincide with the boundaries between domains within the protein encoded by that gene.

2. Many genes, and proteins they encode, have apparently arisen during evolution in part via exon duplication and divergence. 3.Related exons are sometimes found in unrelated genes. ★ Exons have been reused in genes encoding different proteins

Topic 6 : RNA editing RNA editing is another way of changing the sequence of an mRNA. Two mechanisms mediate editing: Site-specific deamination Guide RNA-directed uridine insertion or deletion

Site-specific deamination The process occurs only in certain tissues or cell types and in a regulated manner.

RNA editing by deamination of human apolipoprotein gene

Adenosine deamination also occurs in cells. The enzyme ADAR (adenosine deaminase acting on RNA) convert A into Inosine. Insone can base-pair with C, and this change can alter the sequence of the protein. ★ An ion channel expressed in mammalian brains is the target of Adenosine deamination.

Guide RNA-directed uridine insertion or deletion. determining where the Us will be inserted directing the gRNAs to the region of mRNAs it will edit

Topic 7: mRNA transport Once processed (capped, intron-free and polyadenylated ), mRNA is packaged and exported from the nucleus into the cytoplasm for translation.

How are RNA selection and transport achieved? RNA associates with proteins as soon as transcription : Initially proteins involved in capping, then splicing factors, and finally the proteins that mediate polyadenylation. Some proteins are replaced at various steps during the processing path,but some such as SR are not, moreover,additional proteins join.

As a result, a typical mature mRNA carries a collection of proteins that identifies it as being mRNA destined for transport. Others not only lack the particular signature collection required for transport, also have their collection that block transport.

Examples : The excised introns carry hnRNPs which mark such an RNA for nuclear retention and destruction. Mature mRNA carry residual SR proteins, even another group of proteins binding specifically to exon-exon junctions. Conclusion :the set of proteins, not any individual kind of protein, marks RNAs for either export or retention in the nucleus.

Nuclear pore complex : a specific structure in the nuclear membrane. Small molecules – under 50Kd – can pass through it unaided. And large molecules and complexes require active transport. mRNAs and their associated proteins can actively transport through NPC

Transport of mRNAs out of the nucleus NPC GTPase

The end !!