Small molecule activators of pre-mRNA 3′ cleavage

Abstract

39 Cleavage and polyadenylation are obligatory steps in the biogenesis of most mammalian pre-mRNAs. In vitro reconstitution of the 39 cleavage reaction from human cleavage factors requires high concentrations of creatine phosphate (CP), though how CP activates cleavage is not known. Previously, we proposed that CP might work by competitively inhibiting a cleavagesuppressing serine/threonine (S/T) phosphatase. Here we show that fluoride/EDTA, a general S/T phosphatase inhibitor, activates in vitro cleavage in place of CP. Subsequent testing of inhibitors specific for different S/T phosphatases showed that inhibitors of the PPM family of S/T phosphatases, which includes PP2C, but not the PPP family, which includes PP1, PP2A, and PP2B, activated 39 cleavage in vitro. In particular, NCI 83633, an inhibitor of PP2C, activated extensive 39 cleavage at a concentration 50-fold below that required by fluoride or CP. The testing of structural analogs led to the identification of a more potent compound that activated 39 cleavage at 200 mM. While testing CP analogs to understand the origin of its cleavage activation effect, we found phosphocholine to be a more effective activator than CP. The minimal structural determinants of 39 cleavage activation by phosphocholine were identified. Our results describe a much improved small molecule activator of in vitro pre-mRNA cleavage, identify the molecular determinants of cleavage activation by phosphoamines such as phosphocholine, and suggest that a PPM family phosphatase is involved in the negative regulation of mammalian pre-mRNA 39 cleavage.

Structurally diverse low molecular weight activators of the mammalian pre-mRNA 3′ cleavage reaction

“…A repeat of this experiment is shown in Supplementary Figure S1. Cyclopeptide cp38 is therefore ~1000-fold more potent than the most potent 3′ cleavage activator previously identified, 14 and phosphorylation of its serine, which is required for PPM1 inhibition, 23 is required to activate pre-mRNA 3′ cleavage. PPM1D is present at very low levels in HeLa cells, 27 an observation that leads us to suppose that cp38 is likely not working through this PPM1 family member, but may instead be a family-specific inhibitor that activates 3′ cleavage through another family member, or homologous protein.…”

“…2A) was identified as a 3′ cleavage activator through a limited structure-activity relationship study undertaken using commercially available compounds related to our initial lead, leucine β-naphthylamide, a weak PPM1A inhibitor. 14 To search for a more potent activator than 1 , we synthesized a series of analogs in which the naphthalene ring was altered (Fig. 2A, 2–4 ) or the guanidino group was alkylated or replaced by another positively charged group (Fig.…”

“…Like creatine phosphate, it activates 3′ cleavage over the 10–50 mM range. 14 Its ability to inhibit PPM1 family proteins is unknown. Phosphocholine is an intermediate in the biosynthesis of phosphatidylcholines, and it is unlikely to be a physiological cofactor in 3′ cleavage.…”

“…Phosphocholine is an intermediate in the biosynthesis of phosphatidylcholines, and it is unlikely to be a physiological cofactor in 3′ cleavage. 14 However, understanding the determinants of 3′ cleavage activity of this very simple structure might help us to design more potent activators. To learn whether all three methyl groups are necessary for activity, we synthesized the mono- and dimethyl phosphocholine analogs, 9 and 10 (Scheme 2A).…”

“…PPM1) family inhibitors that stimulated 3′ cleavage in place of creatine phosphate. 14 In humans, the PPM1 superfamily consists of at least eighteen different Mg 2+ - or Mn 2+ -dependent Ser/Thr phosphatases defined by shared sequence homology. 15–18 In plants, the PPM1 family is greatly expanded, with at least 80 family members in Arabidopsis .…”