Outline  Introduction  The chemistry of RNA Splicing  The Splicing Machinery  Splicing Pathway  Alternative Splicing  Exon Shuffling  RNA Editing  mRNA Transport

Introduction  In almost all bacterial and phage genes, the opening-reading frame is a single stretch of condons with no break.  But most of the eukaryotic genes are mosaic, the coding sequences are separated by noncoding sequences

Exons : the coding sequences Introns : the intervening sequences( uncoding streches ) (1)The number of introns found within a gene varies enormously. (2)The sizes of the exons and introns vary..

Critical questions  How are the introns and exons distinguished from each other?  How are introns removed?  How are exons joined with high precision?

※ The primary transcripts (pre-mRNA) must have their introns removed before they can be translated into protein ※ Introns are removed from the pre-mRNA by a process called RNA Splicing. ※ RNA Splicing converts the pre-mRNA into mature messanger RNA.

The chemistry of RNA Splicing

The borders between introns and exons are marked by specific nucleotide sequences within the pre- mRNAs Sequence within the RNA determine where splicing occurs.

Specific nucleotide sequences within the pre-mRNA: 5’splice site : the exon-intron boundary at the 5’ end of the intron(GU) 3’ splice site : the exon-intron boundary at the 3’ end of the intron(AG) Branch point site : an A close to the 3’ end of the intron, which is followed by a polypyrimidine tract (A)

Figure 13-2 The consensus sequences at the intron -exon boundary

The intron is removed in a Form called a Lariat ( 套索 ) as the Flanking Exons are joined Two successive transesterification reactions

The OH of the conserved A attacks the phosphoryl group of the conserved G. Exon is released and the 5’-end of the intron forms a three-way junction structure. ↖ Three-way junction ○

The structure of three-way function

※ The newly liberated 3`OH of the 5`exon attacks the phosphoryl group at the 3`splice site. ※ The newly liberated intron form a lariat.

○ ○ ○ ◇ ◇ Overview

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.  The Difference: A Y-shaped branch structure is formed instead of a lariat.

Trans-splicing Y-shaped branch ○

THE SPLICESOME MACHINERY

 RNA Splicing is carried out by a large complex called the spliceosome  The spliceosome comprises about 150 proteins and 5 snRNAs It is believed that the RNA components rather than the proteins carry out the functions of the spliceosome.

Components involved: snRNAs: The five RNAs (U1, U2, U4, U5, and U6) are called small nuclear RNAs. snRNPs:The complexes of snRNA and proteins are called small nuclear ribonuclear proteins. Other proteins.

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

Some RNA-RNA hybrids formed during the splicing reaction

Splicing pathways

Assembly, rearrangement, and catalysis within the spliceosome: the splicing pathway  Assembly pathway: Formation of early (E) complex Formation of A complex Formation of B complex  Rearrangement: Formation of C complex  Catalysis: Formation of the active site

Formation of E complex: 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 ← E complex ←A complex U2 binds to the branch site, and then A complex is formed

U4, U5 and U6 form the tri- snRNP Particle With the entry of the tri- snRNP, the A complex is converted into the B complex. Tri- snRNP particle ↗ B 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

 Formation of the C complex produces the active site.  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.  U5 snRNP helps to bring the two exons together, and aids the second transesterification reaction  Release of the mRNA product and the snRNPs

E complex A complex B complex Overview of splicesome -mediated splicing reactions C complex Tri- snRNP particle

Self-splicing introns reveal that RNA can catalyze RNA splicing  Self-splicing introns: the intron itself folds into a specific conformation within the precursor RNA and catalyzes the chemistry of its own release and the exon ligation.  Practical definition for self-splicing introns: the introns that can remove themselves from RNAs in the test tube in the absence of any proteins or other RNAs.

There are two classes of self-splicing introns, group I and group II self- splicing introns in eukaryotes. They are not enzymes, because they mediate only one round of RNA prodessing.

TABLE 13-1 Three class of RNA Splicing ClassAbundanceMechanismCatalytic Machinery Nuclear pre- mRNA Very common; used for most eukaryotic genes Two transesterificatio n reactions; branch site A Major spliceosome Group II introns Rare; some eu- Karyotic genes from organelles and prokaryotes Same as pre- mRNA RNA enzyme encoded by intron (ribozyme) Group I introns Rare; nuclear rRNA in some eukaryotics, organlle genes, and a few prokaryotic genes Two transesterific- ation reactions; exogenous G Same as group II introns

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

Group Ⅰ Introns Group Ⅰ Introns release a linear rather a lariat. They use a free G nucleotide or nucleoside instead of a branch site A residue. The two-step transesterification reactions are the same as that of splicing of the group II intron and pre-mRNA introns.

Three forms of splicing

How does spliceosome find the splice sites reliably ？ The active site of the spliceosome is only formed on RNA sequences that pass the test of being recognized by multiple elements during spliceosome assembly. Yet, the problem of appropriate splice-site recognition in the pre-mRNA remains formidable: It seems inevitable that many errors would occur

Two kinds of splice-site recognition errors:  Splice sites can be skipped.  “Pseudo” splice sites could be mistakenly recognized, particularly the 3’ splice site.

Errors produced by mistakes in splice-site selection

Two ways in which the accuracy of splice-site selection can be enhanced : First, it assembles on the sites soon after they have been synthesized. Second, there are other proteins---SR proteins--- that bind near legitimate splice sites and help recruit the splicing machinery to those sites. So-called SR proteins bind to sequences called exonic splicing enhancers(ESEs)

SR proteins, bound to exonic splicing enhancers (ESEs), interact with components of splicing machinery, recruiting them to the nearby splice sites.

Alternative Splicing

Single genes can produce multiple products by alternative splicing. Exons can be extended,or skipped, also, introns can be retained in some messages.

Single genes can produce multiple products by alternative splicing. Drosophila DSCAM gene can produces different mRNA and proteins.

Exons can be extended,or skipped, also, introns can be retained in some messages.

The previous mechanisms described that exons are not skipped and splice sits not ignored. So, how does alternative splicing occur so often? The answer is that some splice sites are used only some of the time,leading to the production of different versions of the RNA from different transcriptions of the same gene.

There are two forms of alternative splicing : Regulated alternative splicing: different forms are generated at different times, under different conditions, or in different cell or tissue types. Constitutive alternative splicing: more than one product is always made from the transcribed gene. (See picture for details)

Constitutive alternative splicing in the troponin T antigen (generating two different T- ag )

Alternative splicing is regulated by activators and repressors Proteins regulating splicing bind to specific sites called Exonic (or intronic) splicing enhancers ( ESE or ISE) or silencers ( ESS or ISS ) The former enhance and the latter repress splicing. Proteins that regulate splicing bind to these specific sites for their actions.

Repressors and activators are respond for alternative splicing, As a result, a particular exon is chosen in a certain cell.

Alternative splicing as a way in which multiple protein product can be produced from a single gene, these different proteins are called isoforms. Some gene that encodes only a single functional protein also show alternative splicing. Why??? As a way of switching expression of the gene on and off (by two pathway)

Inhibition of splicing by hnRNPI Binds at each end of the exon and conceals it. Coats the entire Exon.

A small group of intron are spliced by minor spliceosome This spliceosome works on a minority of exons, and those have distinct splice-site sequence. Using a low- abundance form of splicing. The chemical pathway is the same as the major spliceosome. (Read page 400 for more details)

Exon Shuffling

Exons are shuffled by recombination to produce gene encoding new proteins Why introns are rare — almost nonexistent —in bacteria? Are they lost or never existent? Why does it happen?

Two Models Introns early model – introns existed in all organisms but have been lost from bacteria. (Bacteria become gene- rich responding to selective pressure) Intron late model – introns never existed in bacteria but rather arose later in evolution.

Advantages of the presence of introns: First, the presence of introns, and the need to remove them, allow for alternative splicing which can generate multiple proteins from a single gene. Second, having the coding sequences of genes divided into several exons allow new genes to be created by reshuffling exons.

The evidence of  The borders between exons and introns within a given gene often coincide with the boundaries between domains within the protein encoded that gene.  The borders between exons and introns within a given gene often coincide with the boundaries between domains within the protein encoded by that gene.  Many genes, and the proteins they encode, have apparently arisen during evolution in part via exon duplication and divergence.

RNA Editing

RNA editing is another way of changing the sequence of an mRNA  RNA Editing is another mechanism that allows an RNA to be changed after transcription so as to encode a different protein from that encoded by the gene.  There are two mechanisms that mediate editing: Site-specific deamination Guide RNA-directed uridine insertion or deletion

A tissue- specific manner by deamination of a specific cytodine to generate a uridine.

Guide RNA-directed uridine insertion or deletion. ※ Multiple Us are inserted into specific region of mRNAs after transcription (or US may be deleted). ※ This form of RNA editing is found in the mitochondria of trypanosomes. ※ Us are inserted into the message by so- called guide RNAs(gRNAs).

Three regions of gRNA editing region – determining exactly where the Us will be inserted anchor– directing the gRNAs to the region of mRNAs it will edit. poly-U stretch ↑ ↑↑ ↑ ↑

RNA ligase → RNA-DNA duplex with looped out single strand regions →

RNA Transcription

Once processed, mRNA is packaged and exported from the nucleus into the cytoplasm for translation All the fully processed mRNAs are transported to the cytoplasm for translation into proteins. So, how are RNA selected and transport achieved???

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.

※ Critical concepts: exon, intron, RNA splicing, trans- splicing, self-splicing, RNA editing ※ The chemistry and pathway of RNA splicing ※ The ways to enhance accuracy ※ The mechanism of selecting RNA to be transported ※ Understanding ways to increase diversity of proteins generated by the same gene: (1)Alternative splicing (most important of all) (2)Exon shuffling (3)RNA editing (more economic?) Summary