Cuaternario (Q)

5.1 INTRODUCTION

In c h a p te r 4 seq u en ce e le m e n ts in v o lv e d in re g u la tin g gene tran scrip tio n was discussed. A fter the m essage is tran scrib ed it un d erg o es sev eral p o st-tran scrip tio n a l p ro cesses. T h ese in clu d e rem oval o f in tro n s, addition o f the cap stru ctu re to the 5' end and the poly-A tail to the 3' end, tran sp o rt to the cy to p lasm , translation and finally degradation o f the m essage. R egulation of th e m R N A s ta b ility and th u s th e m e s sa g e a v a ila b le fo r tra n s la tio n p ro v id e a c o n sid e ra b le d e g re e o f fle x ib ility in m an ip u latin g gene ex p ressio n , as sm all changes in tran scrip tio n can be am plified through altered degradation o f the m essage.

Several elem ents, both cis-elem en ts and trans actin g facto rs are in v o lv e d in re g u la tin g m R N A sta b ility (p a rtly d isc u sse d in ch ap ter 3).

5.2 The role o f the poly-A tail in reg u latin g m R N A stability in som atic cells.

O ne of the im portant elem ents is the poly-A tail, w hich has been show n to reg u la te tran sla tio n a ctiv a tio n o f d e ad e n y la te d la te n t tPA m RN A during oocyte m aturation. In som atic cells how ever, d ead en y latio n o f the poly-A tail usually leads to d eg rad atio n of the m essage. E v id en ce for this p ro cess has been ob tain ed by sev eral groups of w orkers in clu d in g N u d el e t al. (19 7 6 ) w ho show ed th at ra b b it 6 -g lo b in m RN A w ith a ta il o f 32 p o ly -A

n u cleo tid es has the sam e fu n ctio n al stab ility as a m essage w ith 150 nu cleo tid es. W hen the tail is d ead en y lated to 16 resid u es the stab ility o f the m RN A d ecreases by 10-fold or clo se to the fully dead en y lated state. G enerally m essages w ith less than 30 n u cleo tid es in th eir poly-A tail are rare. W h eth er at this tail le n g th th e d e a d e n y la tio n sto p s o r th e tr a n s c r ip ts u n d e rg o im m e d ia te d e g ra d atio n is y et to be e lu c id a te d , b u t th is d a ta suggests th at deadenylated m R N A s are generally unstable. T hat d e ad e n y la tio n is the p relim in ary e v en t o f m essag e d e g ra d atio n has b een d e m o n stra te d fo r sev eral tra n sc rip ts in c lu d in g c-fo s and c-m yc (W ilson and Treism an, 1988).

T h e c lo s e lin k b e tw e e n p o ly -A re m o v a l a n d m e s s a g e d eg rad atio n , the existence o f specific signals fo r cleav ag e, poly- a d en y latio n , and d ead en y latio n su g g est th at th is is a reg u la te d ev en t m o d u lated th ro u g h tran s-a ctin g fac to rs. A p ro te in th at bind to the poly-A tail w as first d em o n strated by Sachs et al. (1986). The pro tein term ed the poly-A b inding protein (PA B P) has been c h arac terise d and found to be re m a rk ab ly co n serv ed from yeasts to hum ans (Sachs and D avis, 1989). It is a protein of 577 and 633 am ino acids in yeast and hum an resp ectiv ely . The p ro tein consists o f tw o dom ains. T he N -term in al dom ain w hich has hom ology to o th er RNA binding p ro tein s is th o u g h t to be im portant for RNA binding function. T his dom ain has also been p roposed to be resp o n sib le for the ab ility o f PA B P to m igrate from one poly-A to another. The C -term inal dom ain is thought to in teract w ith other cytoplasm ic facto rs th at reg u la te m essage s ta b ility .

E xperim ents th at have estab lish ed PA B P as hav in g an essen tial fu nction in m RNA stability in clu d e w ork by B rew er and R oss (1989), w ho found PA B P could reg u late d eg rad atio n o f B-globin m RNA s possessing poly-A tails but not those lacking a tail.

W h ile c o m p e ti t io n e x p e r im e n ts w ith 6- g lo b in m R N A dem onstrated that there is a 7-fold rise in its degradation in the presence o f com petitor poly-A polym ers, but n o t in the presence o f poly-C , poly-G or poly-U sequences (B ern stein et al., 1989a, 1989b) T hese data im ply that binding o f PA B P p ro tects poly-A tails from d ead en y latio n , p o ssib ly th ro u g h sh ield in g from being attacked by poly-A nucleases.

A lthough PA B P has a role in m RNA stability it is not the only t r a n s a c t i n g fa c to r th a t is in v o lv e d in re g u la tin g m e ssa g e d eg rad atio n as d ifferen t m essages can be d eg rad ed at d ifferen t rates. T herefore m RN A degradation m ay inv o lv e PA B P as w ell as o th er m ech an ism s w hich act th ro u g h o th e r sig n als in the m RNA m olecule.

5.3 T h e function o f A and U n u cleo tid e rich elem en ts in RN A d e g r a d a tio n

T he A U -rich sequences are know n to m ediate m essag e stab ility (b rie fly d isc u sse d in c h a p te r 3). T he A U U U A p e n ta m e r is p resent in the 3’ UTR of m any unstable transcripts such as c-fos, c-m yc and G M -C SF (g ran u lo cy te m acro p h ag e-co lo n y stim u latin g fa c to r) and has been show n to im p o se in s ta b ility to th o se m essages carrying it.

W ilson and T riesm an (1988) show ed deletion o f the 3' A RE from c-fos leads to the m utant m RNA becom ing m ore stable than the short lived w ild-type c-fos mRNA. W hilst transfer of the G M -CSF A RE containing several AU UU A elem ents to the 3' U TR o f the m ore stable 6-globin m essage leads to a decrease in the 6-globin h alf-life from 17 hours to 30 m ins. An id en tical c o n stru ct but w ith d isru p ted A U U U A s does n o t d epress the 6-g lobin h a lf-life as m uch. In vitro t r a n s c r i p t i o n a s s a y s s h o w e d t h a t tran scrip tio n does n o t account fo r this effect as both constructs h ad s im ila r tra n s c rip tio n ra te s (S h aw an d K a m en , 1 9 8 6 ). F u r th e r m o r e B re w e r an d R o ss (1 9 8 8 ) d e m o n s tr a te d th a t en d o n u clease cleavage o f c-m yc m RNA takes p lace im m ediately 3’ of its AUUUA element.

5 .4 Trans acting factors that bind to A U -rich seq u en ces.

S e v e ra l A U b in d in g fa c to rs h a v e now b e en id e n tifie d by d ifferen t w orkers varying in size from 30 to 70 k D a . M alter (1 9 8 9 ) id e n tifie d a p ro te in co m p lex (A U B F ) th a t b in d s to o lig o n u c le o tid e s w ith 4 A U U U A s, b u t n o t to id e n tic a l o lig o n u c le o tid e s la c k in g th e p e n ta m e rs o r c o n ta in in g th e in v erted sequence U A A A A U . V akalapoulou et al. (1991) have identified a 32 kD a nuclear protein in H eLa cells that can bind to the ARE o f GM -CSF 3' UTR, w hile Brew er (1991) using an in vitro assay sy stem have id en tifie d a tran s-a ctin g fa c to r (A U -F ) th at bind to the c-m yc ARE and accelerate its turnover.

F u rth e rm o re , B o h ja n e n e t.a l. (1 9 9 1 a ,b ) fo u n d th a t T c e lls sy n th esize tw o clo sely related cy to p lasm ic facto rs term ed AU-B and AU-C that specifically bind to AUUUA pentam ers o f GM -CSF,

IL-2 and T N Fa but not to the AU element of c-myc.

Several o f the factors, though id en tified in d ifferen t cell system s have sim ilar m olecular w eights and binds to id en tical sequences suggesting th at they m ay be sim ilar fac to rs o r b elo n g to the sam e fam ily . E v id en ce su g g est th a t A U -A (B o h jan en e t al. 1991a), A U B P (V ak alap o u lo u e t al. 1991) and A U B F (M alter 1989) m ay be id e n tic a l fa c to rs. A ll are fo u n d ab u n d an tly , p e rh a p s c o n stitu tiv e ly e x p re ssed and a ll re c o g n ise sim ila r 3' U T R s (tab le 5.1). T here are h o w ev er d ifferen c e s w ith in this group, AUBP and AU-A are predom inantly nuclear w hile A U B F is c y to p la s m ic .

Some o f these factors (eg AUBP) can bind not only to reiterated AUUUA elem ents, but also to a single pentam er when it is in a U- rich co n tex t (V ak alap o u lo u 1991, B rew er 1991) w hich su g g est th at the w id er A U -rich reg io n has an im p o rta n t ro le in the binding o f these proteins to 3' UTRs.

R ecent evidence indicates th at som e o f these facto rs are related to th e h e te ro g e n e o u s n u c le a r r ib o n u c le a r p r o te in s (h n R N P ). T h ese are a fa m ily o f a b u n d a n t n u c le a r p ro te in s th a t are involved in RNA m etabolism at the level o f pre-R N A processing. All o f these proteins contain RN A binding dom ains (D reyfuss et al, 1988). A 38 kD a protein associated w ith cytoplasm ic poly-A m RNA in HeLa cells has been identified as the hnR N P A1 protein. T h is p ro te in w as show n to be ab le to sh u ttle b etw een the n u cleu s and the cy to p lasm w ith in creased accu m u latio n in the cytoplasm after transcription inhibition (D reyfuss et al., 1984).

T a b l e 5.1: S e v e r a l of t h e A U - r i c h e l e m e n t bi n di n g p r o t e i n s * vD AU bindi ng p r o t e i n S o u r c e S i z e kDa C e l l u l a r l o c a t i o n Binding mRNA R e f e r e n c e

1. AUBF Human p er i phe ra l

blood m o n o n u c l e a r c e l l s 3 6 c y t o p l a s m i c IL, GM-CSF, TNF, v - my c , c - f o s . Mal ter, 1 98 9 . 2. AU-A Human T - l y m p h o c y t e s 3 4 p r e d o mi n a n t l y n uc l e a r IL2, GM-CSF, c - m y c , TNFa Bohj annen, 1 9 9 1 , 1 9 9 2 . 3. AU-B 4. AU-C " " 3 0 . 4 3 , , c y t o p l a s m i c - IL2, GM-CSF, ‘ TNFa, but not

c - m y c " "

5. AUBP HeLa c e l l s 3 2 n u c l e a r and

c y t o p l a s m i c

GM-CSF,