Chapter 13 RNA splicing

Many eukaryotic genes are split. Exons are any region retained in a mature RNA. Introns are intervening sequences removed by RNA splicing.

The average number of introns per gene is larger in higher organisms than in lower ones. The number of introns ranges from 1 to as many as 363. The average size of exons: 150 nt The size of introns: about 3,000 nt, up to 800,000 nt (800 kb) The primary transcript (or pre-mRNA) can be very long because of introns. Ex) human distrophin gene : 2,400 kb /40 nt/sec = 17 hours of transcription RNA splicing should be precise. Without precision of splicing, the reading frame can be changed. Some pre-mRNA can be spliced in more than one way, producing isoforms. Up to 75% of the human genes are estimated to be spliced by alternative splicing. Ex) fly DSCAM gene: more than 38, 000 possible products

THE CHEMISTRY OF RNA SPLICING Sequences within the RNA determine where splicing occurs The borders between exons and introns are marked by specific sequences. 5’ splice site at the 5’ end of the intron 3’ splice site at the 3’ end of the intron branch point site close to the 3’ end of the intron followed by a polypyrimidine tract

The intron is removed in a form called a lariat as the flanking exons are joined An intron is removed by two successive transesterification reactions. In transesterification reactions, phosphodiester linkages within the pre-mRNA are broken and new ones are formed. 2’-OH of A at the branch site attacks phosphoryl group of G at the 5’ splice site 3’-OH of the 5’ exon attacks phosphoryl group at the 3’ splice site Nucleophlilic attack, S N 2 reaction The liberated intron has a shape of a lariat. No net gain in the number of chemical bonds – no energy input is required. But ATP is consumed to assemble and operate the splicing machinery. What ensures the direction of splicing reaction? 1. an increase in entropy, 2. rapid degradation of excised introns

Exons from different RNA molecules can be fused by trans-splicing Trans-splicing of 2 different RNA molecules in trypanosomes and C. elegans

The Spliceosome Machinery RNA splicing is carried out by a large complex called the spliceosome The spliceosome comprises about 150 proteins and 5 snRNAs, and is similar in size to a ribosome. It uses several molecules of ATP for a single splicing reaction. RNA participates in catalysis of the splicing reaction itself. Small nuclear RNAs (snRNAs) are nt long and are complexed with proteins, forming small nuclear ribonuclear proteins (snRNPs) The exact makeup of the spliceosome differs at different stages. RNA-RNA interactions during splicing reactions (Fig. 13-9) Involvement of U2AF, BBP, RNA annealing factors, and DEAD box helicases

SPLICING PATHWAYS Assembly, rearrangements, and catalysis within the spliceosome: the splicing pathway U1 snRNP binds to the 5’ splice site. U2AF binds to the pyrimidine tract and the 3’ splice site, and recruits BBP. U2 snRNP replaces BBP. U2AF leaves the complex, and U4, U5, and U6 snRNPs join the complex. U1 leaves the complex, and U6 replaces it at the 5’ splice site. U4 leaves the complex, and the RNA components of U2 and U6 form the active site. Late formation of active site: Lessens the chance of aberrant splicing

Self-splicing introns reveal that RNA can catalyze RNA splicing Three classes of splicing: nuclear pre-mRNA splicing, group I and group II self-splicing In self-splicing, the intron folds into a specific conformation within the precursor RNA and catalyzes its own release. No proteins are required in the test tube. Mediate only one round of RNA processing: not a true enzyme In the case of group II introns, the chemistry of splicing is the same as that for nuclear pre-mRNAs.

Group I introns release a linear intron rather than a lariat Group I introns use a free G nucleotide or nucleoside. They share a conserved secondary structure. They have G nucleotide-binding pocket & internal guide sequence that base-pairs with the 5’ splice site sequence. Proteins help stabilize correct structure. – in vitro, high salt concentrations were used to reduce repulsion between negative charges of the backbone Group II-like self-splicing introns were the starting point for the evolution of pre-mRNA splicing. The requirement of sequence specificity of introns is provided in trans, instead of in cis. Similarity in folding of the RNA catalytic regions for group II introns & pre-mRNAs

How does the spliceosome find the splice site reliably? Splice site selection is stringent. Splice sites are recognized by multiple elements during spliceosome assembly. Selection of correct splice sites from many splice sites seems to be a difficult task. Splice site recognition is prone to two kinds of errors: exon skipping and use of pseudo splice site Two ways to enhance the accuracy of splice-site selection 1.Co-transcriptional loading: factors bound to the 5’ site are poised to interact with the factors binding to the next 3’ site 2.SR (serine arginine –rich) proteins bind to Exonic Splicing Enhancers and recruit the splicing machinery. They ensure that splice sites close to exons are recognized preferentially. SR proteins not only ensure the accuracy and efficiency of constitutive splicing, but also regulate alternative splicing.

A small group of introns are spliced by an alternative splicesome composed of a different set of snRNPs Some pre-mRNA in higher eukaryotes are spliced by the AT-AC (minor) spliceosome. Minor spliceosome contains unique components: U11 and U12, U4 and U6 equivalents. Recognizes AT-AC and GT-AG termini with consensus sequences distinct from those for major spliceosome. Uses the same chemical pathway Evolution pathway Group II intron  AT-AC (minor)  major pre-mRNA

ALTERNATIVE SPLICING Single genes can produce multiple products by alternative splicing 75% of human genes undergo alternative splicing. In alternative splicing, exons can skipped or extended. Introns can be retained. Alternative exons can be selected.

Alternative exons

5 ways to splice RNA

Alternative splicing can be either constitutive or regulated. In the constitutive splicing, more than one product is always made from the transcribed gene. In the regulated splicing, different forms are generated in a regulated manner. SV40 large T and small t antigens: large T induces transformation and cell cycle entry, whereas small t blocks apoptosis. The more SF2/ASF (by binding to exon 2), the more small t atigen.

Several mechanisms exist to ensure mutually exclusive splicing The most common form of alternative splicing is one with alternative exons. Mutually exclusive splicing caused by steric hindrance occurs when the splice sites within the intron are too close together ex) alpha troponin

Combination of major and minor splice sites Human JNK1 is an example of this kind of mutually exclusive splicing

Nonsense-mediated decay Messages that have one or another exon (never both and never neither) survive from nonsense-mediated decay because they can avoid premature termination

The curious case of the Drosophila Dscam gene: mutually exclusive splicing on a grand scale The Dscam (Down syndrome cell-adhesion molecule) gene potentially encodes 38,016 isoforms, which are cell surface proteins of immunoglobulin superfamily. The Dscam protein establishes neural networks in the brain by mediating specific cell- cell interactions (between axons and dendrites). It recognizes antigens as a part of the innate immune system, much as vertebrate antibodies do.

Mutually exclusive splicing of Dscam exon 6 cannot be accounted for by any standard mechanism and instead uses a novel strategy The novel mechanism is based on the formation of RNA-RNA base-paired structure within the pre-mRNA. Sequence analysis reveals that there are two classes of conserved elements, the docking sites and the selector sequence.

Because selector-binding regions in the docking sites overlap, the binding of different selector sequences to the docking site is mutually exclusive. The binding of the selector to the docking site protects that particular exon 6 variant from a general repression of splicing. Hrp36 functions as a general repressor of splicing by coating the other exon varients.

Alternative splicing is regulated by activators and repressors Splicing factors bind to exonic (or intronic) splicing enhancers (ESE or ISE) or silencers (ESS and ISS) to regulate splicing.

Splicing enhancers are recognized by SR proteins. 1. RNA recognition motif (RRM) to bind RNA 2. The RS domain mediates interactions between the SR protein and proteins within the splicing machinery, recruiting that machinery to a nearby site. Ex) fly Half-pint protein recruits U2AF Splicing silencers are recognized by hnRNPs. They lack the RS domains and blocks specific splice sites. Ex) hnRNPA1 binds an ESS within an exon of HIV TAT pre-mRNA and blocks binding of the activator SC35 to nearby ESE indirectly. SF2/ASF can overcome this repression because it has a higher binding affinity than SC35.

repression of splicing by hnRNPI hnRNPI binds to the Py tract and inhibits binding of the splicing machinery. In other cases, it excludes a given exon by a looping out mechanism or by coating the whole exon through the cooperative binding.

Regulation of alternative splicing determines the sex of flies The sex of a fly depends on which of the two alternative splicing variants of double-sex mRNA it produces. The activator SisA and SisB genes are on X chromosome, whereas the repressor Dpn gene is on one of the autosomes. Sex-lethal gene is regulated by SisA/B and Dpn. Pe (promoter for establishment) is active only in females. Pm (promoter for maintenance) is costitutive in both males and females, but the RNA from Pm has one more exon than RNA form Pe, producing no functional sxl protein. Sxl protein is a splicing repressor that make the inhibitory exon spliced out. In the presence of Sxl protein, functional Tra protein can be produced in females. Tra protein is a splicing activator and regulates Doublesex (Dsx) gene.

EXON SHUFFLING Exons are shuffled by recombination to produce genes encoding new proteins In introns early model, introns existed in all organisms, but had been lost in bacteria in response to selective pressure to increase the rate of replication and cell division. In introns late model, introns were inserted later by a transposon-like mechanism. What are the advantages of the presence of introns and splicing? Alternative splicing generates multiple products from a single gene New genes can be created by exon shuffling 1. Each exon often encodes an independently folding unit. 2. Many genes (and their products) have arisen via exon duplication and divergence. Ex) immunoglobulins

3. Related exons are sometimes found in otherwise unrelated genes. Exons are reused in genes encoding different proteins. Intron/exon size ratio ensures recombination within the introns. Alternative splicing allows new exons to be tried.

RNA EDITING RNA editing is another way of altering the sequence of an mRNA Site-specific deamination of adenines or cytosines In the mammalian apolipoprotein-B gene, Gln(CAA) is converted to STOP(UAA) in a tissue-specific manner by cytidine deaminase. Messages are edited in intestinal cells but not in liver cells. The enzyme contains an RNA binding domain that helps recognize the specific site. ADAR (adenosine deaminase acting on RNA) converts A to I. A single amino acid change in an ion channel in mammalian brains alters the Ca++ permeability of the channel. Without this editing, brain development is severely impaired.

RNA EDITING Guide RNAs direct the insertion and deletion of uridines In RNA editing in mitochondria of trypanosomes, mutiple Us are inserted into specific regions of mRNAs after transcription. Guide RNAs (gRNAs) can be divided three regions: 5’ anchor, the editing region, and 3’ polyU stretch. The editing region is complementary to the region to be edited but contains additional As. After the gRNA and mRNA form an RNA-RNA hybrid, the looped out regions are recognized by an endonuclease.

mRNA TRANSPORT Once processed, mRNA is packaged and exported from the nucleus into the cytoplasm for translation How to select and transport mRNA: a regulated process, not a passive one A typical mature mRNA carries a collection of proteins that identifies it as being mRNA destined for transport. Other RNAs not only lack the particular signature collection of proteins, but have proteins that actively block RNA export. Ex) introns bound by hnRNPs Export takes place through the nuclear pore complex.

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