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RNA SPLICING AND PROCESSING

CHAPTER 21 (GENES X)

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Introduction to RNA processingTranscription elongation is tightly coupled to RNA processing.

Eukaryotic mRNAs are modified at their beginning, middle and end

Why modify mRNA?

Assess if mRNA is intact

Provides a regulatory mechanism for the amount of proteinproduced by a gene

Introns-different forms of a protein from the same gene

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I. mRNA processing (eukaryotes)

1. 5´ capping

2. 3´ cleavage and polyadenylation

3. RNA splicing (nuclear pre-mRNA)

II. Nuclear pore complex overview

III. rRNA and tRNA processing overview

Introduction to RNA processing

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mRNA processing

• Occurs in eukaryotes (but a few bacterial cases exist)

• Primary RNA polymerase II transcripts (pre-mRNA) are usually not functional

• RNA is processed following (or during) transcription

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3 steps of mRNA processing occurs in the nucleus

5’ CAP

Poly-A tail

Splicing

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Eukaryotic mRNA processing overview

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The 5’Cap

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CAP-THIS IS the 5’ end ofan mRNA!

Marks mRNA for export tocytoplasm for translation

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The capping reaction is performed by three enzymes acting in succession:

A phosphatase: removes one phosphate from the 5’ end

of the nacent RNA,

A guanyl transferase: adds GMP in a reverse linkage (5’

to 5’ instead of 5’ to 3’),

A methyl transferase: adds the methly group to the

guanosine.

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5´ capping

• 5´ end of pre-mRNA is covalently modified

• 7-methylguanosine is added

• Linked 5´ to 5´

• Occurs shortly after initiation

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Function of 5´ cap

• Protection from degradation

• Increased translational efficiency

• Transport to cytoplasm

• Splicing of first exon

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Do all RNA polymerases CAP?

Pol I and III do not have the CTD, and do not captheir RNAs

The cap distinguishes mRNAs

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Structure of two human genes showing the arrangement of exons and introns.

(A) The relatively small b-globin gene, which encodes one of the subunits of the oxygen-carrying protein hemoglobin, contains 3 exons (see also Figure 4–7). (B) The much larger Factor VIII gene contains 26 exons; it codes for a protein (Factor VIII) that functions

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Variation in intron and exon lengths in the human, worm, and fly genomes.

(A)Size distribution of exons.

(B) Size distribution of introns. Note that exon length is much more uniform than intron length.

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Splicing

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Introduction to RNA processing

I. mRNA processing (eukaryotes)

3. RNA splicing (nuclear pre-mRNA)

A. Introns, exons, and splicing

B. Spliceosome and snRNAs

C. Self-splicing

D. Alternative splicing

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RNA SplicingRNA Splicing

Primary transcripts (in eukaryotes) are sometimes “spliced” to remove non-coding regions “introns” from coding regions “exons”

The exon regions are spliced together to form the mature mRNA

5’ Cap- - Poly A Tail

hnRNA

Splicing

5’ Cap- - Poly A Tail

Mature mRNA

addition of cap, polyA tail

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Types of mRNA SplicingTypes of mRNA Splicing

Types I & II: self-splicing of catalytic RNA sequences (Ribozymes)

- Two types reflect different ribozyme types

Type III: occurs in a protein - RNA complex - Responsible for nearly all splicing

- It has been speculated that the RNA component of this structure is the catalytic component which would also make it a ribozyme

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Prevalence Of SplicingPrevalence Of Splicing

mRNA: Eukaryotes - most mRNAs in vertebrates- many mRNAs in invertebrates- some mRNAs in unicellular

eukaryotes

Type III:

Type I & II:Eukaryotes - rRNA in tetrahymena nuclei

(Type I)- mRNA & rRNA in fungal mitochondria (I & II)- mRNA in some chloroplasts (II)

Prokaryotes - mRNA in bacteriophage T4 (I)

no type III mRNA splicingin Prokaryotes!

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Mechanism of pre-mRNA Splicing(Spliceosome Mediated) Type III

Mechanism of pre-mRNA Splicing(Spliceosome Mediated) Type III

- Introns have conserved sequences at the splice junctions

- SnRNPs (Small nuclear ribonuclear proteins) binds critical sites on the pre-mRNA

- pronounced ‘SNURPs’

- these are complexes containing both protein and small RNAs

- the small RNAs are transcribed by RNA polymerase III

- they then associate with accessory proteins

- the complex then recognizes critical sites for splicing by base pairing

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The consensus nucleotide sequences in an RNA molecule that signal the beginning and the end of most introns in humans. Only the three blocks of nucleotide sequences shown are required to remove an intron sequence; the rest of the intron can be occupied by any nucleotide. Here A, G, U, and C are the standard RNA nucleotides; R stands for either A or G; Y stands for either C or U. The A highlighted in red forms the branch point of the lariat produced by splicing. Only the GU at the start of the intron and the AG at its end are invariant nucleotides in the splicing consensus sequences. The remaining positions (even the branch point A) can be occupied by a variety of nucleotides, although the indicated nucleotides are preferred. The distances along the RNA between the three splicing consensus sequences are highly variable; however, the distance between the branch point and 3' splice junction is typically much shorter than that between the 5' splice junction and the branch point.

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Conservation of sequence is expected with recognition of sequences being done by base pairing with snRNP’s RNA component

- consensus sequences are conserved throughout eukaryotes

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The RNA splicing reaction.

(A) In the first step, a specific adenine nucleotide in the intron sequence (indicated in red) attacks the 5' splice site and cuts the sugar-phosphate backbone of the RNA at this point. The cut 5' end of the intron becomes covalently linked to the adenine nucleotide, as shown in detail in (B), thereby creating a loop in the RNA molecule. thereby creating a loop in the RNA molecule. The released free 3'-OH end of the exon sequence then reacts with the start of the next exon sequence, joining the two exons together and releasing the intron sequence in the shape of a lariat. The two exon sequences thereby become joined into a continuous coding sequence; the released intron sequence is degraded in due course.

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Excised intron sequence in form of a lariant structure

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Nuclear pre-mRNA splicing

• Introns: non-coding sequences

• Exons: coding sequences

• RNA splicing: removal of introns and joining of exons

• Splicing mechanism must be precise to maintain open reading frame

• Catalyzed by spliceosome (RNA + protein)

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Classes of intronic splicing: splicesome

• Major pathway for splicing and is used for processing of all pre-mRNAs in eukaryotes

• Splicesome is a complex of five small nuclear ribonucleoprotein particles (snRNPs).

• Each snRNPs consists of a small nuclear RNAs (snRNA) associated specifically with proteins.

• Large, ~60S (size of a ribosome), 5 snRNAs (U1, U2, U4, U5, U6) and roughly 50 proteins!

• Splice at specific markers with the mRNA using the snRNA for recognition and catalysis!

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Once all of the different snRNPs associate with their appropriate targets on the pre-mRNA

the entire (very large) complex is called a:

Spliceosome

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Splicesomal mechanism

• snRNA U1 has sequences that are complementary to 5’ splice site

– U1 plus its protein component is the U1 snRNP

• upon U1 snRNP binding, U2, U4, U5 and U6 snRNP assemble

– ATP is required for assembly but not catalysis! (needed for RNA helicases that allow alternative pairing schemes)

Proteins removed from this view for clarity!

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Secondary structure model of human U1 snRNP. The region where it recognizes the pre-mRNA is also shown

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Important point is recognition and complementarity of 5’ splice site sequences with U1 RNA sequences.

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• The E complex contains:– U1 snRNP bound at the 5′ splice

site– the protein U2AF bound to a

pyrimidine tract between the branch site and the 3′ splice site

– SR proteins connecting U1 snRNP to U2AF

Figure 26.10

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Splicesomal mechanism

ATP is required for assembly but not for catalysis

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The RNA splicing mechanism.

RNA splicing is catalyzed by an assembly of snRNPs (shown as colored circles) plus other proteins (most of which are not shown), which together constitute the spliceosome. The spliceosome recognizes the splicing signals on a pre-mRNA molecule, brings the two ends of the intron together, and provides the enzymatic activity for the two reaction steps (see Figure 6–26). The branch-point site is first recognized by the BBP (branch-point binding protein) and U2AF, a helper protein. In the next steps, the U2 snRNP displaces BBP and U2AF and forms base pairs with the branch- point site consensus sequence, and the U1 snRNP forms base-pairs with the 5' splice junction (see Figure 6–30). At this point, the U4/U6∑U5 “triple” snRNP enters the spliceosome.

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In this triple snRNP, the U4 and U6 snRNAs are held firmly together by base-pair interactions and the U5 snRNP is more loosely associated. Several RNA–RNA rearrangements then occur that break apart the U4/U6 base pairs (as shown, the U4 snRNP is ejected from the splicesome before splicing is complete) and allow the U6 snRNP to displace U1 at the 5' splice junction (see Figure 6–30). Subsequent rearrangements create the active site of the spliceosome and position the appropriate portions of the pre-mRNA substrate for the splicing reaction to occur. Although not shown in the figure, each splicing event requires additional proteins, some of which hydrolyze ATP and promote the RNA–RNA rearrangements.

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Doublechecking of intron boundary sequencesSeveral of the rearrangements that take place in the spliceosome during pre-mRNA splicing.Shown here are the details for the yeast Saccharomyces cerevisiae, in which the nucleotide sequences involved are slightly different from those in human cells. (A) The exchange of U1 snRNP for U6 snRNP occurs before the first phosphoryl-transfer reaction (see Figure 6–29). This exchange allows the 5' splice site to be read by two different snRNPs, thereby increasing the accuracy of 5' splice site selection by the spliceosome. (B) The branch-point site is first recognized by BBP and subsequently by U2 snRNP; as in (A), this “check and recheck” strategy provides increased accuracy of site selection. The binding of U2 to the branch-point forces the appropriate adenine (in red) to be unpaired and thereby activates it for the attack on the 5' splice site (see Figure 6–29). This, in combination with recognition by BBP, is the way in which the spliceosome accurately chooses the adenine that is ultimately to form the branch point.

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(C) After the first phosphoryl-transfer reaction (left) has occurred, U5 snRNP undergoes a rearrangement that brings the two exons into close proximity for the second phosphoryl-transfer reaction (right). The snRNAs both position the reactants and provide (either all or in part) the catalytic site for the two reactions. The U5 snRNP is present in the spliceosome before this rearrangement occurs; for clarity it has been omitted from the left panel. All of the RNA–RNA rearrangements shown in this figure (as well as others that occur in the spliceosome but are not shown) require the participation of additional proteins and ATP hydrolysis.

U5 is present here

U5 snRNP undergoesrearrangement

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The commitment (E) complex forms by successive addition of U1 SnRNP to the 5’ splice site, U2AF to the pyrimidine track/3’ splice site, and the bridging protein SF1/BBP.

The E complex forms by interactions involving both splice sites.

• ASF/SF2 (a general splicing factor in the SR class)

• U2AF splicing factor (member of SR, arg/ser rich proteins)

U2AF65 contacts pyrimidine track

U2AF35 contacts dinucleotide AG, 3’ splice site.

• SF1 splicing factor connects U2AF to U1 snRNP bound to 5’ splice site.

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Introns ends can be recognized by either of two pathways.

Intron definition Exon definition

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A splicesome forms via several complex.

E complex: Formation of commitentComplex in which U1 is basepaired with the 5’ splice site

A complex: U2 addition to basepair with the branch site in the presence of ATP

B1 complex: Joining of U4.6/U5 tri-snRNPs

B2 complex: U1 and U4 releaseFormation of the catalytic center in which U6 basepairs with U2;U2 reamins basepaired with the branch site; U5 interacts with both exons through its loop.

C1 complex: The first step of transesterification5’ splice site cleaved, lariant formed

C2 complex: The second step of transesterification3’ splice site cleaved, exaons ligated

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The exon definition hypothesis. According to one proposal, SR proteins bind to each exon sequence in the pre-mRNA and thereby help to guide the snRNPs to the proper intron/exon boundaries. This demarcation of exons by the SR proteins occurs co-transcriptionally, beginning at the CBC (cap-binding complex) at the 5' end. As indicated, the intron sequences in the pre- mRNA, which can be extremely long, are packaged into hnRNP (heterogeneous nuclear ribonucleoprotein) complexes that compact them into more manageable structures and perhaps mask cryptic splice sites. Each hnRNP complex forms a particle approximately twice the diameter of a nucleosome, and the core is composed of a set of at least eight different proteins. It has been proposed that hnRNP proteins preferentially associate with intron sequences and that this preference also helps the spliceosome distinguish introns from exons. However, as shown, at least some hnRNP proteins may bind to exon sequences but their role, if any, in exon definition has yet to be established.

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3´ cleavage and polyadenylation

• RNA polymerase II does not usually terminate at distinct site

• Pre-mRNA is cleaved ~20 nucleotides downstream of polyadenylation signal (AAUAAA)

• ~200 AMPs are then added to the 3´ end

• Almost all mRNAs have poly(A) tail

The 3’ end of mRNA is generated by cleavage. mRNA is stabilized by polyadenylation.

• The sequence AAUAAA is a signal for cleavage to generate a 3′ end of mRNA that is polyadenylated.

• The reaction requires a protein complex that contains:– a specificity factor– an endonuclease– poly(A) polymerase

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• The specificity factor and endonuclease cleave RNA downstream of AAUAAA.

• The specificity factor and poly(A) polymerase add 200 A ∼residues processively to the 3′ end.

• A-U-rich sequences in the 3’ tail control cytoplasmic polyadenylation or deadenylation during Xenopus embryonic development.

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Consensus nucleotide sequences that direct cleavage and polyadenylation to form the 3' end of a eucaryotic mRNA.These sequences are encoded in the genome and are recognized by specific proteins after they are transcribed into RNA. The hexamer AAUAAA is bound by CPSF, the GU-rich element beyond the cleavage site by CstF (see Figure 6–38), and the CA sequence by a third factor required for the cleavage step. Like other consensus nucleotide sequences discussed in this chapter, the sequences shown in the figure represent a variety of individual cleavage and polyadenylation signals.

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Basic steps of poly-Adenylation

• Most eukaryotic mRNA have 80-250 Ade added to their 3’ end by a multi-step process.

• CTD of Pol II has a 7aa (x52) that strongly stimulates polyadenylation

– Endonuclease (complex) cleaves 10-30 nt downstream of AUAAAA

– PABPI recruits polyA pol

– Poly-A pol adds A in association with PABPII

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3’ modification-addition of a poly A tail

CSTF- cleavage stimulation factor

CPSF- cleavage and polyadenylation specificity factor

PAP- PolyA polymerase

PABP- Poly A binding proteins

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3’ modification-addition of a poly A tail

CSTF- cleavage stimulation factor

CPSF- cleavage and polyadenylation specificity factor

PAP- PolyA polymerase

PABP- Poly A binding proteins

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Some of the major steps in generating the 3' end of a eukaryotic mRNA.

This process is much more complicated than the analogous process in bacteria, where the RNA polymerase simply

stops at a termination signal and releases both the 3' end of its transcript and the DNA template.

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Function of poly(A) tail

• Increased mRNA stabilityProtects mRNA In cytoplasm poly(A) size decreases due to RNAses, however poly(A) polymerase continues rebuilding: No poly(A) tail…destroyed!

• Increased translational efficiencyNecessary for translation (site of binding by poly(A)-binding protein I (PABI) Recruits mRNA to polylysomes so that translation can be initiated

• Splicing of the last intron

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Degradation of RNA

• mRNAs are degraded at different rate (control of gene expression)

• Life time varies from several seconds to several generations. T1/2= 3hours for vertebrate and 1.5minutes for bacteria.

• RNA is degraded from 5’ to 3’. • 3’-sequence often inhibit 3’-5’

exoribonuleases.

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Are you ready for export?

CappA tailNo snRNPs boundOther proteins bind thatIndicate mRNA is ready

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Nuclear transport

• Nucleus houses DNA and is site of transcription and RNA processing

• Cytoplasm is site of translation

• RNAs must leave nucleus

• Some proteins must enter nucleus

• Nucleus is separated from cytoplasm by nuclear envelope (double biomembrane)

• Transport across nuclear envelope is through numerous nuclear pores

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Nuclear pore complex

• Portal through nuclear envelope

• Mediates traffic in and out of nucleus

• Very large multiprotein complex

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THE NUCLEAR PORE COMPLEX (NPC) I

1. 125 x 106 D, spans the inner and outer membranes of the NE

2. Octagonal symmetry

3. Represents the transit path for molecules going into and out of the nucleus

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Transport of a large mRNA molecule through the nuclear pore complex.

(A) The maturation of a Balbiani Ring mRNA molecule as it is synthesized by RNA polymerase and packaged by a variety of nuclear proteins. This drawing of unusually abundant RNA produced by an insect cell is based on EM micrographs such as that shown in (B).

61The EJC (Exon Junction Complex) binds to RNA by recognizing the splicing complex.

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Transcript export

Proteins associated with mRNA mark it for

export

Only mature mRNA is exported from nucleus

Exit via nuclear pore complexes

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A REF protein binds to a splicing factor and remains with the spliced RNA product. REF binds to an export factor that binds to the nuclear pore.

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Schematic illustration of an “export- ready” mRNA molecule and its transport through the nuclear pore. As indicated, some proteins travel with the mRNA as it moves through the pore, whereas others remain in the nucleus. Once in the cytoplasm, the mRNA continues to shed previously bound proteins and acquire new ones; these substitutions affect the subsequent translation of the message. Because some are transported with the RNA, the proteins that become bound to an mRNA in the nucleus can influence its subsequent stability and translation in the cytosol. RNA export factors, shown in the nucleus, play an active role in transporting the mRNA to the cytosol (see Figure 12–16). Some are deposited at exon-exon boundaries as splicing is completed, thus signifying those regions of the RNA that have been properly spliced.

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1. Get at gene through chromatin2. Pol II recruited to bind promoter via a host of TFs (up and down)3. Rearrangement of PIC to start elongation 4. Capping (cotranscriptionally!)5. Splicing (+ alt splicing) (cotranscriptionally!)6. Termination polyadenylation7. Export to cytoplasm8. Translation9. turnover

Putting it all together

All of these steps are regulated and all can contribute to increasing or decreasing the levels of mature mRNA!

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ALTERNATIVE SPLICING MECHANISMS

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ALTERNATIVE SPLICING

A COMMON PRE-mRNA IS SPLICED INTO MULTIPLE

mRNA ISOFORMS DIFFERING IN THEIR COMBINATION

OF EXON SEQUENCES.

THIS GENERATION OF MULTIPLE mRNAS FROM A

SINGLE GENE PARTIALLY UNDERLIES THE APPARENT

DISCREPANCY BETWEEN GENE NUMBER AND THE

COMPLEXITY OF THE ORGANISM.

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Alternate SplicingAlternate SplicingSplicing at alternate splice sites can lead to the production of several gene products from one gene

- Proteins involved in splicing can bring alternate domains of pre-mRNA together to give alternate splicing events

- Alternate splicing can be regulated

- differential splicing can occur at different times during development and in different cell types

1 2 3 4GENE

(hnRNA)

1

1

2

3

3

4

42 Different mRNA products2 Different mRNA products

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• The regulation of RNA splicing can generate different versions of protein in different cell types, according to the needs of cell.

• Tropomyosin for example is produced in specialized forms in different types of cells.

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Example of alternate splicing with rat -tropomyosin Example of alternate splicing with rat -tropomyosin

Alternative splicing of the a-tropomyosin gene from rat. a-tropomyosin is a coiled-coil protein that regulates contraction in muscle cells. The primary transcript can be spliced in different ways, as indicated in the figure, to produce distinct mRNAs, which then give rise to variant proteins. Some of the splicing patterns are specific for certain types of cells. For example, the a- tropomyosin made in striated muscle is different from that made from the same gene in smooth muscle. The arrowheads in the top part of the figure demark the

sites where cleavage and poly-A addition can occur.

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Alternative splicing!

• ~30% of human genes are subject to alternative splicing

• Regulated as a function of cell/developmental stage as well as in response to signals

• Range of exons from 1 to 54 (HGP)– Percent of human DNA in exons 3%– Total number of genes ~33,000 but alternative

splicing increases the diversity of protein

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ALTERNATIVE 5’ SPLICE SITES

ALTERNATIVE 3’ SPLICE SITES

SINGLE ALTERNATIVE EXON:

EITHER INCLUDED OR SKIPPED

MULTIPLE ALTERNATIVE EXONS:

ONE OR OTHER CAN BE CHOSEN

INTRON RETAINED AND TRANSLATED

From BR Graveley, Trends in Genomics:17(2001)100-107

Alternative Splicing Patterns

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Four patterns of alternative splicing: In each case single type of RNA transcript is spliced in two alternative ways to produce two distinct mRNAs (1 and 2). The dardk blue boxes mark on exon sequences that are retained in both mRNAs. The light blue boxes mark possible exon sequences that are included in only one of the mRNAs. The boxes are joined by red lines to indicate where intron sequences (yellow) are removed.

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Negative and positive control of alternative RNA splicing. (a) Negative control, in which a repressor protein binds to the primary RNA transcript in tissue 2, thereby preventing the splicing machinery from removing an intron sequence. (B) positive control, in which the splicing machinery is unable to efficiently remove a particular intron sequence without assistance from an activator protein.

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Alternative Splicing can change a protein’s properties

• Individuals with an X/A ratio of 1 (normally two X chromosomes and two sets of autosomes) develop as females.

• X/A ratio of 0.5 (normally one X chromosomes and two sets of autosomes) develop as males.

• This ratio is assigned early in development and is remembered thereafter by each cell.

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Sex determination in Drosophila

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Sex determination in Drosophila

Three crucial gene products transmit information about this ratio to the many other genes that specify male and female characteristics.

5th edition, Chapter 7: control of gene expression, page 479-483

81A cascade of changes in gene expression that determines the sex of a fly through alternative splicing.

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SEX DETERMINATION IN DROSOPHILA: A CASCADE OF ALTERNATIVE SPLICING

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SECOND STEP OF SPLICING OF SXL PRE-MRNA

SPF45=SECOND STEP SPLICING FACTOR

FUNCTIONSIN MALES

SXL BINDS UPSTREAM OF AG,THEREBY INTERACTING WITH AND INHIBITING SPF45

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ALTERNATIVE SPLICING OF TRA PRE-MRNASXL binds to the pyrimidine-rich stretch of nucleotides that is part of the standard splicing consensus sequence and blocks access by normal splicing factor, U2AF.

FULL-LENGTH TRA PROTEIN

TRUNCATED TRA PROTEIN

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SR proteinSR protein

TRANSCRIPTIONAL REPRESSOR OF FEMALE-SPECIFIC GENES

TRANSCRIPTIONAL REPRESSOR OF MALE-SPECIFIC GENES

MALE

ONLY PRODUCEDIN FEMALES

Tra binds to specific RNA sequence in an exon and with Tra2 activates a normally suboptimal signal by the binding of U2AF. Tra is a positive activator of RNA splicing.

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Here is what an alternative splice might look like on a protein

• Protein may have different ligand specificity or enzyme activity etc…

Splice product 1 Splice product 2

Alternative splicing of Slo gene

• The mammalian cochlea is a snail-like structure of the inner ear that contains hair cells organized along a basilar membrane.

• There are four rows of hair cells, one inner hair cell and three outer hair cells.

• The hair cells are turned to unique narrow sound frequencies along the basilar membrane and creating a tonic gradient.

• At one end the hair cells are tuned to respond to a frequency of 20 Hz, other side cells respond to 20,000 Hz.

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• Hair cells tunning is thought to be mediated, in part, by the alternative splicing of transcripts expressed from the calcium-activated potassium channel gene, Slo.

• Isoforms of the Slo gene lacking sequences encoded by the STREX-exon have fast deactivation kinetics and low ca++ sensitivity,

• where as STREX containing sequences have a slower deactivation kinetics and higher ca++ sensitivity.

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Alternative splicing of Slo gene

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Alternative splicing in human slo gene; >500 possible isoforms.(believed to be involved in hearing sounds of different frequencies.)

From BR Graveley, Trends in Genetics:17(2001)100-107.

Constitutive splicing

Alternative splicing, alternative exons are grey boxes

Isoform of Slo protein lacking sequences encoded by STREX exon

Neuronal signaling and synaptogenesis

• Neurexins are family of a neuronal proteins present in vertebrates that have important functions as receptors for neuropeptides and adhesion molecules that participate in synaptogenesis.

• b-neurexins containing exon 20-encoded sequences can inhibit synaptogenesis, whereas exon-20 containing neurexin does not.

The relative ratio of the two protein isoforms could determine whether functional synapsis are formed or broken.

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Alternative splicing in human neurexin 1 gene; >300 possible isoforms.(believed to be involved in making and breaking synaptic connections between neurons.) From BR Graveley, Trends in Genetics:17(2001)100-107.

The LNS domains of neurexin and agrin undergo alternative splicing that modulates their affinity for protein ligands in a neuron-specific manner.

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ALTERNATIVE SPLICING IN THE NERVOUS SYSTEM:

GENERATION OF MUCH OF THE ENORMOUS DIVERSITY

NEEDED IN THE PROTEINS INVOLVED IN FORMING

SPECIFIC SYNAPTIC CONNECTIONS AND IN MEDIATING

SYNAPTIC TRANSMISSION

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Alternative splicing of Drosophila DSCAM gene: DSCAM proteins are axon guidiance receptors that help to direct growrh cones to their appropriate targets in the developing nervous system. The final mRNA contains 24 exons, four of which (denoted A, B, C and D) are present in DCAM gene as arrays of alternative exons. Each RNA contains 1 of 48 alternative exon A (red), 1 of 48 alternative exon of B (green), 1 of 33 alternative exon of C (yellow). If all possible splicing combinations are used, 38,016 different proteins could be in principle be produced from the gene. Each variant DSCAM protein would fold roughly the same structure. (predominantly a series of extracellular immunoglobulin like domains linked to a mambrane-spaining region) but the amino acid sequence of the domains would vary according to the splicing pattern. It is possible that this receptor deversity contributes to the formation of neural circuits, but the precise properties and functions of the many DSCAM variants are not yet understood.

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DROSOPHILA DSCAM GENE ENCODES AN AXON GUIDANCE

RECEPTOR THAT CAN EXPRESS 38,016 mRNAS VIA ALTERNATIVE

SPLICING

EXON 4 ALTERNATIVE SPLICING IS DEVELOPMENTALLY REGULATED:

EXON 4.2 INFREQUENTLY USED IN EARLY EMBRYOS, PREDOMINANT IN

ADULTS

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Alternative splicing in Dorsophilia Dscam gene; 38,000 possible isoforms.(believed to be involved in Axon migration and connection.)From BR Graveley, Trends in Genetics:17 (2001)100-107.

Neurons expressing the form of Dscam shown on the right will be attracted in a different direction than neurons expressing the form on the left.

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TISSUE-SPECIFIC RNA SPLICING FACTOR IN THE NERVOUS SYSTEM: NOVA-1, A

REGULATOR OF ALTERNATIVE SPLICING IN THE BRAINSTEM AND SPINAL CORD

Nova-1 null mice show specific splicing defects in two inhibitory receptor pre-

mRNAs, glycine alpha2 exon 3A (GlyRalpha2 E3A) and GABA(A) exon gamma2L.

Nova protein in brain extracts specifically bound to a previously identified

GlyRalpha2 intronic (UCAUY)3 Nova target sequence, and Nova-1 acted directly on

this element to increase E3A splicing in cotransfection assays (Neuron. 2000

Feb;25(2):359-71).

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Regulation of the sites of RNA cleavage and poly-A addition determines whether an antibody molecule is secreted or remains membrane-bound. In stimulated B lymphocytes(left), a long RNA transcript is produced, and the intron sequence near its 3’ end is removed by RNA splicing to give rise to an mRNA molecule that codes for a membrane bound antibody molecule. In contrast, after antigen stimulation (right) the primary RNA transcript is cleaved upstream from the spliced site in front of the last exon sequence. As a result, some of the intron sequence that is removed from the long transcript remains as coding sequence in the short transcript. These are the nucleotide sequences that encode the hydrophilic C-terminal portion of the secreted antibody molecule.

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Self-splicing introns

• Introns that splice themselves out of genes

• Ribozymes: catalytic RNA

• Two major types (group I and group II)

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Self-Splicing (Type I)Self-Splicing (Type I)

T.R. Cech, ~1982 made a revolutionary discovery:

RNA could have catalytic activity

RNA enzyme = ‘Ribozyme’

- For this type of splicing, only require:

1. Pre-rRNA

2. Mg2+

3. G (GDP,GMP, guanosine) –guanine does not work

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Self-Splicing Introns fold so as to:

1. Bring 5’ & 3’ splice sites together

2. Form a G-binding pockets close to the splice sites

3. Mg2+ :1. Stabilizes the folded RNA structure

2. Undoubtedly helps to lower G for bond transfer

3’-OH of the {G} cofactor initiates the reaction

- attacks phosphate at the 5’ splice site.

- itself becomes attached to the 5’ end of the intron

- Role equivalent to branch site adenine in type III splicing

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Classes of intronic splicing: Group IFound in some mitochondrial/chloroplast rRNAs, tRNAs and mRNAs

• Requires guanosine as cofactor (not energy)

• 3’ OH acts as nucleophile in the two transesterification reactions

– G attacks 5’ splice site to form 3’-5’ phosphodiester (to 5’ end of intron)

– 3’ OH of displaced exon acts as a nucleophile and attacks the 3’ end of of the intron , yields precise excision of intron!

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Group II self-splicing introns

• Found in plant and fungal organelles

• Mobile DNA elements (nonviral retrotransposons)

• Encode maturases (proteins) needed for physiological splicing

• Similar secondary structure (and splicing mechanism) as snRNAs in spliceosome

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Classes of intronic splicing: Group IIoften in mitochondrial/chloroplast genes in fungi, algae and plants

• Similar to Group I except nucleophile in first transesterification step is 2’ OH of an intronic A

• Forms a “lariat” intermediate from 2’-5’ bond

• again, no energy required

• Like Group I splicing it is entirely dependent on a complex 3D fold of the RNA for activity

104

Predicted secondary structures

105

106

107