Lecture 8 - 10/6

tranitpaionl mode of regualtion is the major mode which cell uses tho controll geen expression. tranciptioal mode by controlling tranciptioal activatio or repssion is the most strongest the cell uses to controll gene xpresion. the cells contain thousands of tranciptioal facotrs called revuatloy tranitpn factors . chagning tf in reposne to signals or by using ocmbibantorl mode of tf expression in dif tissues cell an contrll expression of those genes.

that isnt the only way = nfkb mediated gene expression nfkb being a tf another interest aspect that keepign tf in the cytoplasm the cell can contrll those genes, it isnt jsut increase or decrasing the levels of tf. nfkb by keeping in cytoplasm to not activate tranciption but a specific stress singal can put it in the enhancer. in the next three classes co or post trnaiptioal mode of gene regualtion. once gene is trancribed /syntehies its beocming a protein proudct like protein coding genes. there are other levels or regualtion of that rna molcule.

over view of what we know of post or co tranciption. gene as a dna sequnece, more thatn 90% of the human genes contaon introns, split genes the gene contains introns anc exons and exons make the protein product because they code for amino acids, and introns are interveing sequences that get spliced out. once mRNA gets trancribed few things that hppens on the mRNA . one is that the five prime end of the mRNA gest capped , addiotn of modified nucelotide, a five prim capping and then the three prime end of the trnaciption gets a pola tail that is a strecht of a nucleotiesn taht is up to 150 to 200 and added durign or after the tranciptiton. the a nucelodes is not present on the gene sequence. ther eis not sequence on the gene , this is polAtailing. 5’ cap 3’ poly. the thrid preoces is that the introns are removed/spliced togehter of exons.

pol2 encoded trancitpsn they need to have a cap and poly a because if not the trancipts will be degraded and cannot be exported into the cytoplasm. This is essential . this process can be controlled by reguatlro factors. these 5’ cap 3’ tail stablizes. when gene is trancibed into rna mocluels those trancips are precursor mRNA. until the three process happens it is called mature mRNA.

we are going to focus on the splicing part. every premRNA needs to eb capped, and it is modified Guanin that is added to the 5’ n and this g is not presen on the gene and it is added onto the trancipt. funciton? it stablizes the trancipt/ reason is that normally when two nucelotides are polymersized on a trancipt there is a speicif orentation which the nuelcoeteis will be added onto an exsisting trancipt. what u call polymerization of a trancipt or addion of nuceltodes to make an rna which is nothing but. polymer. the oritentin inwhich each nuceltodes is addes in in a five prime to three prime direction. this particualr nucldoetide which is will be added at the five prime end of the trancipt added by a group of mutli protein enzyme, it connects the 5’ end of trancipt with a G . This modification si five prime to five prime modication. five prime o the g is added to five prime of the trancipt. because of that the ell do not have enzyme that recongize and clave this fieve priem nucleotien. most o fhte ribonucelases that cut rna moleucles dont recgvonze the five -five prime linkage. Becuase of thsi the ribonucelus dont recogvnize and cannot cleave and make the five prime end most stable, if u remove the g the rna will be unstable. the second funciton is that it faciltiates the export of mRNA from ncuelsu to cytopalsm because the mRNA has to tbe in the cytoplasm for tranlationg to happen. there are protei whic specifially recongzie the capped structure and the protien can itneract with nuceloprin compelx to help bring them out into the cytoplas. funciton of cappign also helps tranlation. the caps can recruit ribsomes onto the mRNA.

stablitity

nuclear export

tranlation

impaortnt funcitons of capping

next aspect is the splicign process - if u look at trancipt or preMRA if u look at the mamllian genes, the size of exons are shorter than introns. the average siz eo fexons is 150 and intons are 2-4thousand kb. long introns short exons .exonic sequence are those regions that code for amino acids. every three nucleotide code for amino acids. introns are very degeranted - a lot of mutations happens in introns, because the exon sequences are essentiatl so if any mutation happens on an exon that will have an effect on the protien fucntion and organims survival so mtuation in exons are less tolerated. introns aquire more mtuations and well tolerated. why do we need to keep itnrons?

there are some evidence that introns have evoltionary function but not for the survival of the organims to surivive. introns are important for evolution not immediate funciton of a gene. splicing is removal of introns and combing exons. most o fhte facots that requre foe effeicent splicing is present inside the introns not insde the exons. speicif splicing to happens i sthat the 5 prime. end of the intron there are few sequence at the 3’ end of the introns, there are five prime splice site and three prime splice site are highly conserved, 100 100 is the level of conservated so the first two nuceltides GU of the five prime end of the intron is one hundredd % conserved fro everyitnron. the three prime pslice site whihc is made up of AG is 100 percent conserved. if you go down the next sequnce is 70-80 and goes doens. GU is most important and AG is impoartnt. about twnety to rhtiry nuceltodesupstream of the three prime splice site is a A sequence called branch point a sequence. between the branch poitn there is a sterch of purimadine rich sequence which is required. so intron to plice out, u need conserved five and three pirme splicte cite, a branc h site and a pruamdin renricheds e4quenc ebtween the branch poitn and the prime splci cite.

introns can be 3k nucletodes to 100 kilboases these are so degenrated but still conserved so it nees to be there. GU and AG. this is major spliceosome sites. branch poitn a and pryamidne sequence. tghe

minus spliceosm ecomplex will have a AU branch poitn pruyamin and AC 3’ . its not the major one

splicing is two chem reactions called transesteerifaciotn wher etwo of the bonds are broken and religated back, splic is nothgin but nucleotide is the last nucelar of the exon and first nucelator of the intron and there will be abond broke that is the fist transesteriffaciton reaciton. exon will be related from the his exon will

be released from the 5 prime end of the intron. and

this phi prime end of the intron will turn back

and then form what is called a lariat

structure of the intron, whereas this

exon, which is now free at this point,

will go and go to the next exon and activate

a second transistification reaction, breaks that

bond, which connects at that point the intron

and exon, and basically ligate it back. So

that's what all are called splicing reactions.

the branch point a has a free OH group that activates a transestrifcation/nucleophilic attach which cuts this bondso we have a free exon and 5’ will come back and form a larian and seonc reaciton is thta hydroxyl group of the las nucelsotide activate the next nuclophulic attach and form a lario. two breaking and two ligating back togehter. there are nearly two hndre proiten to work togehter. five small rna moclueslshould fucniton toegehter. te chem reacito in is simple, and u need 200 protiens and fvei small ncuelar RNA, snRNA. together thsese snRNA or usnRNA, which are 100-150 ncueltodeis long, they are unriched with uridine whcih is the nuceltoides. there is u1,u2,u4,u5,u6 there are five of them. u3 is not inovled in splciign of premRNA. this is the splice-usome ocmplex . these faiclate two trancestrifaction reactions.

the five priem splice cite are conserved the reason why is because if u look at the five prime pslice cite GU that sequence can form hydorgen bond with the u1 uSNRNA mocleules . that 5’ splic cite is recognzied by u1snRNA / so they will form a xomplex. it forms a hydrogen bond , the seocn reaciton is the u2 snRNA will from hydrogen bonds with several nucletodes next ot the branch point A sequence. they dont from any interactions at thei point with the 3’ splice cite. if u look atht picture branch point a it is producingt out, it isnt froming h bonds with the u2, its jsut the nuceltoides that are next ot he A. free a is required to activate the ncuelpphilic attack with the 5’ GU area . so the a has to be free. in order of teo chem reaciton to happen branch poitn a has to be very close to the sequenc ehwihc is the frist reaciton . what the snRNA job is to remodigy the rna structure. such that these branch point a can come clso eot the exonic sequence, and active splicign and by holding exons togeth the protien complex wil bind and hold this ushca. way that once the first tranestrificaiotn happens, u dont want hta exon to fly away from that reigon. once exon intron is broke, the rn has no coonnect ot he exon. snRNA will hold it together and keep it togehter os the exon isnt lost. spliceosme is to keep the rna structure modified. There are expiermetn scientst have done to see these itneraciton essential. u can modify the ncueltoides on the snRNA so it can no more bidn to the exon or intron and then the spclign doesnt tka eplace. so u need that hydrogen formation to happen so that snRNA can recongzie thos reigons. so u remove that it cannot activate pslcing. bu scitenst what they have done is called compensatory mtuation expierment wher eu make a mutatnat snrna whihc can no more bind ot the juntion the five prime junciton but now tu matuat ethe mRNA sequence whihc is no complemtaty to the mtuated snRNA so that htey can from hydrogen bond. now the splcign can eb reactivated indicating tha tocmplemttny is moreessentail than the sequence .

during evutalion they use that GU sequencea thte f and AG at the 3 prime because snRNA can have that sequence. thats the basic essential splicitn greaciton, introns are longer how does. the cell specify which is the exon and which is the intron ebcause as far as the nuceltiedes are conced of an mRNA se4quence ther ei sno differentce. its just four ncuelotides in dffereint combinations. so there is nothgin which is aprt of th exonic sequence which will differe from the intronic seq4uence. but ell has to differentat what sexon and whats introns. so i can effeinclty pslice that remove the tinron so that exon can be stitched togethe to active the splcing. so this is very diffuctil to answer.

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this isdone by a group of priten scalled sr family of prietns that are 20 of them.

s and r stands for serine and argiine. So the SR family of preoints all contain RS repeats arginine serine di peptide repeats.

Sr family of preins are essential genes and if u mutate them the cell cannot suvive and htey are splific factos/reguatlor and what they do is they can define the xon sequence so they can identify what an exon sequence is come from intronic squence. how ? Sr family of preoitns , i fu look

athe exonic sequence there is a specif csequence called exonic splicing enahcners callaed ESC that are highly enriched in exonic sequencs. anything that is exon have these seven nuceltoides sequences . Sr pritens tend to bind to exonic sequence bcause of the patterns/nuceltoide squences.

Once sr binds ot eh mtuiple esc’s on exons, thee sr prtoiens cna recuirt snrns to the five prime splice site and three prime splice site so the boundary od the exonic sequence will be the best place where snRN’as will be enrished. other than the middle intronic sequence.

where it is intron there ar eno sr pritne siting here ot recruit ht snRNA. we call thsee cross exon reovnizion complex becaus ethe sr proitns wil recruit uSnRNA to the five prime splice cite and the ohter thre prime facto o fhte intronwhcih is upstream. not the same intron, one exons will recuirt facts oin two ends of the exonic regions.

7 nuceltoide consensous.

one more aspect - rna polymerase trancibed throuhg a gene contirnously and the average speed of a rna pol2 is about two killobases 2thousand nuceltoides within a min when apssting through a gene. cotrantipcioal splicint needs to ahappen as and when the pol is passing throuhg the gene. splicing ahappens at the same time. co troantialaio because pslicing take palce even before the pol is done.

some introns are dont pst and co trantpiaolly, most are co trnatipoal pslicnig meaning at th same time. how doe these factors speicially reocnize trancip taht is coming out of hte gene. if u know the simpel biochem reactions is promoted cirtial concentration of factors in that regions. we need cirital concentraiotn of factors near th geen in order for splciitn to take place.

CTD has high affinity to splicing facots. when rna pol2 is trancibein the ctd tial acts as a platform fo rs plfiicng facots and other factors to bind to that reigon. that way the ractors are present near by the gene. CTD is ysptsps serine two and serine five, serine five is phroyalted by tf2h whih makes pol2 the intation copentnt from. that means that the intaitl prhoaylteion of the serine five is required for pol2 to activate teht ranipttion.

that phroyalted sering five pol2 will recruti the capping enzyme in the intail stages of polymeizaiton reaciton. the capping enzyme also gets recruited to the ctd so the acpaping cna happen at the five priem end of hte tranciption. the capping ithis five prim modified guanosine cap is unqieu featurfre of the pol2 trancipt. pol1,3 do not ctd. so only pol2 has the capping phoryaltion of serine five and new enzyme will active prhoualte of serine two of hte ctd. and that modified ctd si what modified the splicing complex. serin 5 recuits the capping and serin 2 phroylateion recuirt the splic complex. when we see poladylation reaction the same ctd can recruit factors whihc poladneualtion reaction.

that is essntial splicing reatoin, its not taht regualtory. for our classpoint of view . alternatinve splicng. there is consittive splciing where every introns is removed and every exon is ligated togehter . 95% of the genes in our body contain multiple itnrons and mutliple exons but itnerestly that this pcoess what is called alternative splcing seems to be the major player which tornsol the regualtory mechamsi of gene expression. wh

alternative slcing - three exona two introns

every gene starts wiht an exon and ends with an exons . always introns are in the inside .

e i e i e

theoritclaly u can have two dif types of splicing regalure splicing three exons will bind together. but in thsi comination he middle exon can be considered as an intron and be removed form the system . these are isomers. isomere 1 will ahve exons 1,2,3 and isoform will have 1,3 u cannot have 1,2 or 2,3 because u dont have that intronic sequenc ewher eu can activate splciign. inreality this i real situation. trancip whihc is x numbe of exons can have different ocmbiation of splicing comination all coming from the orgianl premRNA that can make dif type os fisomers many of these exons are inclduign es or exloded and could poientally make a domain on a protien. and by removing the odmian the protien cannot fucniton. the domain can be a kinase domain, or it can be acetytltranfease odmain of a proiten. human genom only has 20thousand protien codnign genes and these can make million of cominatoin of preoitns . some genes like in flies called DS-CAM involed in neural fucniton that makes 32thousand isomers from one gene. all thess isomers can be functioal . alternate splicing reactions.

how altenrate pslcing have major impact on fucnitoaly of organism. based on using cancer cells laternive splcing is a ket process that gets dereuglated in cnacner patients, either abonal expression of splicng factors out of those 200 factors we talked about inclduign sr fmaily proiten one o fht ekey oncogenes. just by overexprssion the splicing facot in these patients sampel,

ust by overexpressing the splicing factor

in these patient samples, breast, liver,

multiple cancers, it doesn't matter,

that they can change the splicing potential of

these downstream transcripts, and that will have an

impact on cancer patients. And we have drugs now

being in the, you know, it's coming out, where

we can use to control the splicing factor activity

so that can potentially change the fate of that

particular patient that we are treating, right?

So I'll stop here and then we'll go talk about this

alternate splicing reaction in the next class.