Chapter 4:

This should be later changed to chapter 3

RNA ELEMENTS INVOLVED IN SPLICING

William F. Mueller and Klemens J. Hertel*

Department of Microbiology & Molecular Genetics,

University of California, Irvine,

Irvine, CA 92697-4025, USA

* To whom correspondence should be addressed. E-mail: khertel@uci.edu

due to other chapters: could you mention putative exon silencers (Tito baralle)

1

could you mention ATAC exons, the minor spliceosome (Luhrmann)

explain the ‘splicing code’

2

key concepts

exons and introns are defined by the 5’ and 3’ splice sites, which follow the

degenerate sequences YAG/guragu and yyyyyyyyyyynyag/G

the genome contains 10 times more pseudoexons than authentic exons (can you

double check this number)

exon recognition depends on the interplay of multiple splicing regulatory elements,

that can be intronic or exonic and act as either enhancers or silencer (ESE, ESS, ISE,

ISS,)

pre-mRNA splicing is coupled to transcription and is influenced by promoter type,

polymerase speed, histone modifications, and polyadenylation signals

all these factors contribute to the combinatorial control of splice site selection

The human genome encodes approximately 25,000 genes (IHGSC 2004) [1],[1] and

more than 90% of these are believed shown to produce transcripts that are

alternatively spliced [2-4](Wang et al 2008, Pan et al 2008, Fox[2,3,4] 2009). Isn’t

this number at least 95? Alternative splicing of pre-mRNAs results in the production

of multiple mRNA isoforms from a single pre-mRNA, thus significantly enriching the

proteomic diversity of higher eukaryotic organisms [5, 6](Maniatis and Tasic 2002,

Johnson et al 2003)[5,6]. The regulation of this process can determine when and

where particular mRNA protein isoforms are produced with that have the potential

to modulate various cellular activities.

3

Sequence elements within in the pre-mRNA define the ends of the introns that will

be excised. Exon/intron boundaries are recognized by direct interactions between

the spliceosome and pre-mRNA sequence elements. The formation of the

spliceosome requires the activity of more than 300 distinct protein factors and the

U1, U2, U4, U5, and U6 small nuclear RNAs (snRNAs) [7, 8] need to settle on a

common number(snRNA), [Jurica and Moore 2003, 7,8]). In the classical splicing

model these spliceosomal components assemble onto the pre-mRNA in a stepwise

manner [9] (see chapter 5, Luhrmann). splieceosomal components assemble onto

the pre-mRNA in a stepwise manner (Black 2003).[9] After initial splice-sitesplice

site selection and pairing [(Reed 1996, Lim and Hertel 2004, Kotlajich et al

2009),10,11,12] the catalytic components of the spliceosome are activated and

extensively rearranged, ultimately resulting in intron removal via two trans-

esterification reactions [10-13][13](Staley and Guthrie 1998). There are several

RNA elements that mediate efficient definition of exons and introns. This chapter

will focus on the basic principles that control initial splice site recognition and how

the interplay between various RNA sequence elements results in the generation of

differentially spliced mRNA isoforms.

Splice Site Sequence

The identification of splice sites is the first step in the process of pre-mRNAs

splicing. The 5’ splice site (also referred to as the donor site) is defined as a single

sequence element, 9 nucleotides (nts) in length (Figure 1). In mammals, this site

4

follows a degenerate consensus sequence YAG/GURAGU (where Y is a pyrimidine, R

is A or G, and the / denotes the actual splice site) [14] (Sun and Chasin 2000) that

base pairs with U1 snRNA in early spliceosomal E complex, and with U5 and U6

snRNAs in subsequent complexes. The 3’ splice site (also referred to as the acceptor

site) is defined by three sequence elements that are usually found within 40 nts

upstream of the intron/exon junction. These elements are the branchpoint

sequence (BPS), the polypyrimidine tract (PPT), and the actual 3’ splice site (the

intron/exon junction). The PPT varies in length and is characterized by a high

percentage of pyrimidines. The BPS follows the highly degenerate sequence

YNYURAY (where Y is C or U) flanking a conserved branch point adenosine

(Reviewed Reed 1996[10] [10]). The 3’ splice site is composed of a variable length

PPT followed by the sequence NYAG/G [14, 15]G (Sun and Chasin, 2000, Zhang

1998)[14,15]. U2 snRNP interacts with the BPS, and the PPT functions as a binding

platform for U2 snRNP auxillaryauxiliary factor (U2AF) (Reed 1996).[10] [10].

These RNA elements and their associationed with snRNA /and/or proteins pre-

mRNA and pre-mRNA/protein interactions are essential for initial splice site

recognition. (is it usual to describe the bps as part of the 3’ splice site? Most peope

keep this separate)

The role of these RNA sequence elements is of such importance that the sequence

complementarity of the 5’ splice site to U1 snRNA and the extent of the PPT at the 3’

splice site are used to classify determine the strength of splice sites. Greater

complementarity to U1 snRNA and longer uninterrupted PPTs translate into higher

5

affinity binding sites for spliceosomal components and, thus, more efficient splice

site recognition [16](Hertel 2007)[16]. (however, I think there is no natural 5’

splice site that is fully complementary to U1) Experimental support of this

generalization is abundant. For example, the U1 snRNA complementarity defines

the competitive strength of a 5’ splice site (Roca et al 2005) and 5’ splice sites that

have a high complementarity with U1 snRNA (ie strong 5’ splice sites) splice more

efficiently than those with low complementarity (weak 5’ splice sites) [17] (Hicks

and Shepard, Hertel Lab unpublished data). This concept of complementarity has

been used extensively in numerous methods for the derivingation of numerous

splicing scores [18-20] (Senapathy et al 1990, Zhang and Chasin 2005, Yeo and

Burge 2004). [17,18,19].

The Intron/Exon Architecture

In addition to splice site sequences, their arrangementthe exon/intron architecture

in the pre-mRNA is important for efficient splice site recognition (Berget, 1995).

[21] [20]. In mammals small exons and large introns predominate [9], which

contrasts the situation in yeast Saccharomyces cerevisiae. The average mammalian

exon size is between 50 and 300 nts while the average intron size is 3,400 nts [22].

But for human cassette exon, there is a much sharper distribution around 150 nt?

While the majority of spliceosomal components are conserved between species, the

length and positioning of introns and /exons architecture isare not. The average

mammalian exon size is between 50 and 300 nts while the average intron size is

3,400 nts [21](). In mammals small exons and large introns predominate [9].

6

(Black 2003). This is not the case in Drosophila or yeast where introns are generally

much smaller and exons are larger [23-25] [10,22,23](Reed 1996, Guthrie 1991,

Ruby and Abelson 1991). The variable arrangement and size of exons and introns

suggests that multiple ways of recognizing introns and exons exist, referred to as

intron and exon definition (Figure 2) [21][20]. (Berget 95). Splice site recognition

in the exon definition mode occurs across small exons. The assembled spliceosomal

components then pair across the intron this should be exon to interact with

spliceosomal components associated with a flanking splice site. Intron definition

occurs across small introns where permitting splice site recognition and pairing

within the same intronic splicing unit. Question for reinhard: is it correct to takl

here about spliceosomal components or should it be splicing factors

Experimental support for the intron and exon definition models exist based on

expectations that mutations of exon defined splice sites would result in exon

skipping, whereas mutations of intron defined splice sites would result in intron

retention. These ideas were tested by iIncreasing the size of mammalian exons

tested these ideas, resulting in exon skipping [21, 26][20,24]. (Robberson et al,

1995, Berget, 1995). However, when similar enlarged exons were flanked by small

introns, the exons were included [27](Sterner, 1996[25]). In addition, when splice

sites were mutated from strong to weak, the resulting splicing phenotype was exon

skipping [28, 29](Talerico and Berget 1990, Nakai and Sakamoto, 1994[26,27]).

However, when the length of introns in Drosophila or yeast werelengths of introns

in Drosophila or yeast were increased, intron retention, loss of splicing, and cryptic

7

splicing was observed [30, 31][28,29](Talerico and Berget 1994, Guo et al 1993).

More recent kinetic analyses further showed that weak splice sites are more

efficiently spliced when introns are small [4] [4]. (Fox-Walsh et al 2005). These

results support the concept that splice sites are can be recognized across either the

intron or across the exon.

The above information makes a strong case that the presence of splice sites and the

intron/exon architecture are important for activating pre-mRNA splicing. Still, they

are not the only players. It is known that there are many potential splice sites in the

human genome that are not used and form what are called pseudoexons.

Pseudoexons are unused exons with usable splice sites found in introns or non-

coding regions of pre-mRNAs [14][14]. (Sun and Chasin 2000). Interestingly, they

occur more frequently than true exons by an order of magnitude [32](Zhang et al

2005[30]). Clearly, for pseudoexons to be ignored and for true exons to be

recognized, there must be more information in a pre-mRNA molecule than the splice

site strength and the relative location to adjacent introns and nearby exons. Indeed,

bioinformatic approaches demonstrated that an average region averaging 540 nts

upstream and 80 nts downstream of constitutive splice sitesexons contains

information regarding splice site recognition [33][31]. (Zhang et al 2003). Along

with the remarkable prevalence of pseudoexons, these observations implied that

other regulatory cis-elements exist to help direct the spliceosome to bone fide splice

sites.

8

Splicing Regulatory Elements (SREs)

Once the genome was sequenced, it wasSequencing of the genome verified that the

majority of splice sites did not match the consensus sequence well at all: less than

5% of 5’ splice sites match the consensus and with greater than 25% havinge 3 or

more mismatches from the 9 nt consensus [33][30]. (out of curiosity: is there now a

natural 5” ss that shows 100% match to U1) (Zhang and Chasin 2005). Classical

experiments also demonstrated that exonic sequences outside other than the splice

sites were necessary to correctly process certain transcripts [34][32,33]. (Reed and

Maniatis 1986, ). It was shown that some cis-acting RNA sequence elements

increase exon inclusion by serving as binding sites for the assembly of multi-

component splicing enhancer complexes. These sequence elements, termed exonic

splicing enhancers (ESEs), are were located within regulated exons and were

defined as exonic splicing enchancers (ESEs)[9] [9]. (review Black 2003). Since the

discovery of ESEs other it has been shown that more classes of SREs (splicing

regulatory element) existwere identified. They canSREs recruit proteins and

complexes that can enhance as well as silence splicing and have been named

descriptively: intronic splicing enhancers (ISEs), and exonic and intronic splicing

silencers (ESSs and ISSs). These elements are important for selecting between

pseudoexons and real exons, between competing splice sites, and even for the

splicing of constitutive exons.

ESEs have beenwere identified by their protein binding ability, by analysis of

mutations that decrease splicing efficiency, and by computational comparison of

9

exons. In general, ESEs are recognized by at least one member of the essential

serine/arginine (SR)-rich protein family. These proteins are involved in recruiting

the splicing machinery to splice sites [9, 35].(review Black 2003, Gravely 2000).

I[9,34] It has been proposed that the RS domain of an ESE- bound SR protein

interacts directly with the RS domain of other splicing factors containing an RS

domain, thus facilitating the recruitment of spliceosomal components such as U1

snRNP to the 5’ splice site or U2AF65 to the 3’ splice site [35][34]. An alternative

mode of spliceosomal recruitment was suggested by experiments demonstrating

that RS domains of SR proteins contact the pre-mRNA within the functional

spliceosome [36, 37][35,36]. Irrespective of the RS domain activation mode, SR

proteins facilitate the recruitment of spliceosomal components to the regulated

splice site [9, 38][9,37]. Thus, SR proteins bound to ESEs function as general

activators of exon definition [39][38[] (Figure 3A). Two of the first SR proteins

extensively studied were ASF/SF2 and SC35, which bind ESE sequencess (GAR)n and

GRYYC(G/C)YR respectively [40, 41][(Liu et al 1998, Tacke and Manley 1999,

Cartegni and Krainer 200238,40,41]). These proteins were found to be necessary

for splicing in add back experiments using SR-depleted depleted splicing extracts, as

well as for splice site switching. They have also been shown to be necessary for

splice site choice in multiple instances [42-45][(Ge et al. 199142, Krainer et al.

199143, Fu & Maniatis et al 1990, Zhaler et al 199244,45]). Interestingly, ESE-

dependent SR protein binding sites are present not only within alternatively spliced

exons, but also within the exons of constitutively spliced pre-mRNAsexons [46][46]

(Schaal and Maniatis, 1999). It is therefore expected that SR proteins bind to

10

sequences found in most exons, indicating an extensive role in splicing by ESEs.

Maybe you can mention that there are no SR proteins in cervicia, other than npl3

Not all exon recognition enhancements come from the exonexonic sequences. While

less explored than other SREs, ISEs are vital to many splicing scenarios. For

example, in the alternative splicing of the terminal Calcitonin exon, a conserved

intronic sequence is essential for efficient recognition of the therminal 3’ splice site

(Lou et al 1995site [47][47]). Part of the intronic element is, surprisingly, a cryptic

5’ splice site. Cryptic intronic 5’ splice sites have been shown to act as enhancers in

other contexts [47][(Hastings et al 200148]), however this is not always the case.

Furthermoire, it was observed that recognition of mutually exclusive exons in the -

Ttropomyosin gene requires an ISE that specifically interacts with ASF/SF2 [48]

[49,50]. (Gallego et al (1992, 1997)). These examples, along with those presented

concerning ESEs, support an enhancement- controlled model of splice site

recognition. ESEs canSplicing enhancers activate constitutive, alternative, strong, or

weak splice sites by recruiting SR proteins or splieceosomal components to their

splice sites to enhance exon recognition. Yet that is only half of the picture.

Regulation of pre-mRNA splicing is much more complex than a simple enhancer

recruitment model. Splicing silencers, either ESSs or ISSs, occur frequently and have

been found to influence constitutive and alternative splicing events throughout the

genome [49][51] (Pozzoli and Sironi 2005) (Figure 3B). The best-characterized

silencers are recognized by heterogeneous nuclear

11

ribonuclearproteinsribonucleoprotein (hnRNPs). ISSs usually bindare usually

recognized by the polypyrimidine track binding protein (PTB, also known as hnRNP

I) [50, 51].[(52,53]. Several mechanisms have been proposed for ESS- or ISS-

mediated splicing repression. HnRNP-bound splicing silencers have been shown to

repress spliceosomal assembly through multimerization along exons [52], (Zhu et al

2001),[54] through blocking the recruitment of snRNPs [53, 54](Tange et al 2001,

House & Lynch, 2006)[55,56], or by looping out exons (Martinez-Contreras et al

2006).[55][57]. See chapter 3, Allain for molecular details An interesting finding

regarding silencers is that altering the location of an enhancer can change its

enhancement effect to a silencing effect (Kano pka et al 1996, McNally and McNally

1996[58,59]). Recent advances in identifying splicing silencers have come from

ligand selection/evolution experiments (SELEX [56]) paired with simple kinetic

analysis [57][60]. . It was shown that splicing silencers can alter the U1 binding at

5’ splice sites and that alterations in silencing kinetics can affect splice site choice

[58][61]. (Yu et al 200).

Typically, silencers and enhancers are present within the vicinity of exon/intron

junctions, suggesting that the interplay between activation and repression of cis-

acting elements modulates the probability of exon inclusion. In addition to the

enhancement mentioned previously, studies of Survival of Motor Neuron (SMN) pre-

mRNA splicing have uncovered a number of enhancing and silencing elements

within exon 7 and its flanking introns (Lorson and Adrophony 2000, Cartegni and

Krainer 2002, Kashima and Manley 2003, Singh et al 2004)[41, 59-61]. See chapter

12

17 Singh for a description of the disease[62, 41,, 63, 64]. This suggests interplay

between SREs, most likely influenced by the concentrations of the various splicing

factors involved and the timing of their interactions with the pre-mRNA. I Using in

vitro studies showed , it was shown that the location and frequency of SREs along

the pre-mRNA alter their effectiveness. For example, as the distance between

enhancer complexes and the splice site increased, the probability of exon inclusion

decreased [62][65](Graveley et al. (1998)). Increasing the number of ESEs seemed

to lessen this effect. However, by creating artificial exons with a variable number

and order of ESEs and ESSs, it was shown that the number quantity of enhancers or

silencers had a weak linear relationship with splicing efficiency [63][66]. (Zhang et

al (2009). This conclusion was mainly based on the observation that constructs

with the same ESE to ESS ratios, but different orders of enhancers and silencers

displayed drastically different splicing efficiencies. These results support the notion

that the recognition of most splice sites are influenced by multiple distinct cis-acting

RNA elements and that their activity depends on their context within the pre-mRNA

molecule [16, 63, 64][66,67,16].(Zhang and Chasin 2004, Wang et al 2004. Hertel 0).

RNA Secondary Structure

Single- stranded RNA is likely to adopt secondary folds and tertiary interactions that

may involve up to hundreds of nucleotides. Although pre-mRNAs are typically

depicted in a linear fashion, we have to assume that there exist higher order

structures exist that engage a good portion of the RNA in base pairing interactions.

Depending on the thermodynamic stability or protein binding events, these

13

structures may persist long enough to interfere with or modulate splice site

recognition. Because the recognition of splice sites, enhancers, and silencers usually

depends on interactions between protein factors and a single stranded portion of

the pre-mRNA, In principle, these local structures can inhibit or activate

spliceosomal assembly (Figure 4). This is because the recognition of splice sites,

enhancers, and silencers usually depends on interactions between protein factors

and a single stranded portion of the pre-mRNA [65] (Figure 4)[68]. (). Local RNA

structures can interfere with spliceosomal assembly if they conceal splice sites or

enhancer binding sites within stable helices [66]. On the other hand, local RNA

structures couldan also promote spliceosomal assembly by masking splicing

repressor binding sites.

The importance of RNA secondary structure in modulating splice site selection has

been frequently documented. Can you mention here that dcam can make xx

thousand splice variants For example, the Drosophila Dscam exon 6 cluster has two

classes of conserved RNA elements: a common docking site and a selector sequence

unique to each of the mutually exclusive 48 exon 6 variants. Each selector sequence

can base pair with the docking site to form a secondary structure across 1000 to

14,000 nts, activating and directing mutually exclusive exon pairing (Graveley

2005). [67][69]. An inhibitory role of RNA secondary structure was demonstrated

for SMN2 exon 7 splice -site recognition of SMN2 exon 7. The formation of an RNA

hairpin close to the 5’ splice site of SMN2 exon 7 interfered with its interaction with

U1 snRNP, resulting in reduced exon inclusion levels [68, 69]. (Singh et al 2007).

14

[70,71] In agreement with these observations are computational analyses showing

that ~15% of alternative splicing events strongly correlate with the presence of

stable RNA secondary structures overlapping splice sites (Shepard and Hertel 2008)

[66][72]. These examples support the idea that RNA secondary structures, from

short hairpins to long-range interactions, play a more significant role in modulating

splice -site recognition than perhaps currently appreciated.

Coupling between transcription and RNA processing

It was noted early on that RNA processing events appear to be linked temporally

and physically [70](Kornblihtt et al 2004, Peterson and Perry 1989[73,74]). More

recent studies have demonstrated that the process of splice site recognition can

occur co-transcriptionally [71-73](Gornemann et al 2005, Kornblihtt 2005, Tardiff

et al 2006).[75,76,77]. That is, the splice sites of an exon can be identified and

removed by the spliceosome while downstream exons still await their synthesis by

RNA polymerase II (Pol II). Thus, like 5’ capping and 3’ polyadenylation, intron

removal is linked to RNA transcription.

Transcription of pre-mRNAs is initiated at unique promoter sequences that recruit

Pol II and the rest of theentire transcriptional machinery [74](Levine & Tijan 2003).

[78]. Intriguingly, the alternative splicing pattern of a reporter gene changed

depending on the Pol II promoter identity from which the transcript originated [75]

(Cramer et al 1997)[79]. These results suggested a model in which splicing factors

associate with Pol II close to the promoter. As a consequence, differences in

15

promoter structure could lead to differences in the splicing factors recruited to the

transcription machinery [76](de la Mata and Kornblihtt 200)[80].. An alternative

model proposes that the kinetics of transcription influence alternative splicing. As

Pol II polymerizes in a strict 5’-3’ direction, alternative exons are made prior to or

after the synthesis of competing neighboring exons. Accordingly, the relative timing

of producing competing exons can bring about changes in the splicing pattern.

Strong support for the kinetic proposal stems from experiments testing the effects

of increased intron length, different classes of transcription activators, and Pol II

elongation mutants [76, 77][81,82](,). These kinetic fluctuations during

transcriptional elongation may even be related to a splicing dependent sequence

(not clear what you mean: stucture of the DNA, splicing-dependent change in

histone modification?) context, as recent studies demonstrated that histone

modifications in the vicinity of exons are significantly different from non-exonic

regions [78][83]. ( Related studies showed that alternative splicing decisions

during the processing of human c-Src and Fibronectin pre-mRNAs were made co-

transcriptionally (Pandya-Jones and Black (2009[79][84]). Theses investigations

also suggested that alternative splicing events, such as exon skipping, produce a

change in the kinetics of splicing. , similar to what has been observed when assaying

splicing in vitro. Thus, in addition to the transcriptional efficiency and the act of

alternative splicing may further alter the kinetics of RNA processingefficiency

causing kinetic changes that could alter splice site choice, the act of alternative

splicing may further alter the kinetics of RNA processing, adding even more

dimensionality complexity to an already complexintricate problem.

16

As transcription is approaches termination, 3’ end processing of the pre-mRNA must

occur to protect the pre-mRNA molecule. It was shown early on that mutations of

the terminal intron 3’ splice site or of mutations within the polyadenylation signal

sequences decreased terminal intron splicing efficiency [80-82][74,(Peterson and

Perry 1989, Niwa and Berget, 1990, Niwa et al, 1991)85,86,87],. These observations

argue for a two-way communication between polyadenylation factors and the

spliceosome. Investigations into the regulation of Calcitonin pre-mRNA alternative

splicing demonstrated that a conserved ISE was necessary for the regulation of both

exon 4 recognition and polyadenylation efficiency. Interestingly, an out- of- place 5’

splice site and a PPT found with in exon 4 itself were shown to be necessary for

polyadenylation of the alternative mRNA isoform [47, 83](Lou and Gagel, 1998[88]).

These observations demonstrate interdependencies between splicing regulatory

sequences, alternative splicing, and alternative polyadenylation. Recent genome-

wide analyses revealed that over half of the? human genes have multiple

polyadenylation sites and approximately 20% of these sites are intronic sites that

could lead to mutant alternative variants [84][89]. (Tian et al 2007). In

combination with the extensive use of alternative promoters and alternative

splicing throughout the genome, the coupling of gene expression processes allows

for immensely diverse combinations of pre-mRNA regulation, mainly directed by

the cis-elements found within the pre-mRNA molecule itself.

Combinatorial Effects of Splicing Elements

17

Over the last few years it has become increasingly clear that exon selection is

influenced by a number of activating and inhibitory elements. Given the divergent

sequences and architectures of genes, every exon has its a specific set of identity

elements that permit its recognition by the spliceosome (Figure 5). Each exon is

flanked by a unique pair of splice-sitesplice site signals and contains a unique group

of splicing enhancers, silencers, and secondary structures. Each gene contains

unique promoters and polyadenylation sites that influence the pre-mRNA

processing machinery. The sum of the contributions from each of these identity

elements defines the overall recognition potential of an exon, or the overall binding

affinity for the spliceosome. Considering the variation in splice sites, exon/intron

architecture, the number of enhancers, silencers, and secondary structures, the

recognition potential of exons is expected to span a wide range. The spectrum of

exon recognition potential ranges from exons that are constitutively spliced and

always included in the final transcript to exons that are rarely included or

purposefully excluded from the mature mRNA. The center of the spectrum

represents alternative exons that are sometimes included into the final mRNA

isoform, alternative exons. Both extreme exon classes are expected to be

impervious simpler word resistant?towards subtle changes in the splicing

environment, because their affinity for the spliceosome is so great strong? or poor

that minor changes will not significantly alter overall exon definition efficiencies.

On the other hand, the exon class within the center of the recognition spectrum is

expected to be most sensitive to even minor changes in splicing efficiency. For

example, a small drop in the concentrations of the spliceosomal components or an

18

SR protein could trigger a change from preferential inclusion of an exon to

preferential exclusion. In addition to this, the recent high throughput sequence data

finding that greater than 90% of transcripts are alternatively spliced (Wang et al

2008, Pan et al 2008)[2,3] changes the definition of a constitutive exon. In place of

being a set splicing isoform, this data suggests that a constitutively spliced exon is

simply one that has a significantly higher isoform ratio than another alternative

transcript. This implies that constant RNA surveillance already occurring i

n a cell and might also be coupled to splicing.

When looking at the regulation of a single exon, the effects of influential cis-elements

grow daunting rather quickly. An example of this is the frequently studied inclusion

or exclusion of the constitutive exon 7 in the Survival of Motor Neuron (SMN) mRNA

[69]. Can you move the SMA description up to page 10, where you first mention it?

Two almost identical genes code for the proteins SMN1 (functional) and SMN2

(mostly non-functional). Due to a single base transition, exon 7 is preferentially

skipped in SMN2, resulting in the production of a non-functional gene product

(Figure 6). When the SNM1 gene is mutated or deleted, SMN2 cannot compensate

for its loss and the manifestation of the disease phenotype of Spinal Muscular

Atrophy (SMA) is observed (Cartegni and Krainer 2002[41][41]). Except for a C to U

transition at position 6, exon 7 of SMN1 and SMN2 are identical to each other, each

harboring a weak 5’ splice site. In the SMN1 transcript, that the C at position 6 is

included in a proposed ASF/SF2 enhancer binding site promoting exon 7 inclusion

(Singh 200[69][71]7). In SMN2, the C to U transition is proposed to create an

19

hnRNP A1 binding site that favors exclusion of exon 7. The transition is also

proposed to stabilize an RNA secondary structure that further silences exon

inclusion through extending an inhibitory context [69],[85](Singh et al 2007,

Kashima et al 2007)[71,90]. Outside of the transition region, a conserved ESE

found in the middle region of the exon in addition toand multiple ISSs outside of the

exon add to the growing list of RNA elements involved in mediating exon 7 inclusion

[69, 86][(Kashima et al 2006, Singh 200771,91]). While the combinatorial

regulation of exon 7 is slowly being unraveled, the continued discovery of more

regulatory elements and their interactions shows the complexity of splicing

regulation and, thus, the need for further study.

Even in the face of all this regulation, one large fundamental question remains:

wWhat are the most important parameters for exon recognition? The current

bioinformatic estimation programs only have a 50% accuracy of identifying

correctly spliced exons even with our current knowledge of splicing [87] this is a

fairly old reference I will ask Mike zhang, chapter 46[92]. (Smith and Valcarcel

2000). Although not tested systematically, it is likely that the strength of the splice

sites and their relative proximity are the most crucial aspects of efficient splicing.

Because the spliceosome assembles around splice sites, the binding potential of

splice sites builds the foundation for efficient exon definition. The contributions of

the other parameters will vary significantly from case to case, augmenting the

overall affinity of the splicing machinery. As a consequence of fluctuating

concentrations of spliceosomal components and splicing effectors interacting with

20

the described RNA elements, it is anticipated that the same exon may display

variable exon recognition potentials. Variable exon recognition would increase

between different cell types or between distinct biological processe s. As a result,

exons that are alternatively included in one situation can be alternatively excluded

in another, creating cellular diversity in the organism.

Acknowledgements

We are grateful to the Hertel laboratory for helpful comments on the manuscript.

Our research is supported by NIH grant GM 62287 (K.J.H.)

21

Figure Legends

Figure 1: Consensus sequences of splice sites. A. Diagram of the 5’ splice site. The

5’ end of the intron, recognized initially by U1 snRNP, is identified by the 9 nt

consensus sequence shown. Strong base pairing between U1 snRNA and the 5’

splice site increases the likelihood that a splice site is used. B. Diagram of the 3’

splice site. The three elements within approximately 40 nts of the intron’s 3’ end

are shown here. Because the spliceosomal protein U2AF binds preferentially to

pyrimidines, the longer the stretch of uninterrupted pyrimidines in the PPT, the

more likely the 3’ splice site will be used.

Can you include here a matrix/histogram of the splice sites, can you make a diagram

that shows all elements, similar to fig 6 (ISE, ESE, sec structure, maybe chromatin?,

plus pseudoexon and crytic splice site)

Figure 2: Splice site definition across the intron or exon. A. When the intron is

small (less than approximately 250 nts) the spliceosome can recognize the splice

sites that will be paired across the intron, referring to the intron definition model of

splice site recognition. B. When introns are large (greater than approximately 250

nts), splice sites are recognized across the exon. Because splice sites that will be

paired have been identified separately (on different exons), an additional step is

required to assemble the spliceosome across the intron. This initial definition of

splice sites is referred to as exon definition.

22

Figure 3: SREs in action. A. Enhancement of splicing by ESEs and ISEs occurs

through recruitment of splicing activators that bind to specific RNA sequence

elements, thereby recruiting spliceosomal components to nearby splice sites. B.

ESSs and ISSs are RNA elements that bind splicing repressors thereby inhibiting or

blocking the recruitment of the spliceosomal machinery. The prevalence of SREs

throughout spliced transcripts suggests that they play an extensive role in pre-

mRNA splicing.

Figure 4: The role of RNA secondary structure in splicing. RNA secondary

structures can activate splicing by sequestering silencers within stem loops (left), or

silence splicing by concealing a splice site or enhancer sequences (right) within a

hairpin. Many alternative splicing events appear to correlate with stable RNA

secondary structures [66], although the full extent of local and long range

interactions is unknown.

Figure 5: Summary of cis-acting RNA elements controlling splice site recognition

and exon inclusion. The splice site sequence, intron/exon architecture, RNA

secondary structure, enhancer and silencer sequences, as well as links between pre-

mRNA splicing and transcription or polyadenylation are highlighted. These splicing

elements act in concert to mediate the complex regulation of pre-mRNA splicing.

Figure 6: RNA sequence elements identified in the regulation of SMN pre-mRNA

splicing. The recognition of SMN exon 7 relies on complex interactions between

23

splicing regulatory elements. In this case, a single base change can alter the balance

of enhancers and silencers resulting in different exon 7 inclusion levels.

Combinatorial control of exon definition has been proposed to be required for the

majority of exons due to the widespread occurrence of cis-acting RNA elements

within and surrounding exons, including constitutively spliced exons.

24

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30

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32

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