Reversible inhibition of the second step of splicing suggests a possible role of zinc in the second step of splicing

Abstract

A multicomponent complex of proteins and RNA is assembled on the newly synthesized pre-mRNA to form the spliceosome. This complex catalyzes a two-step transesterification reaction required to remove the introns and ligate the exons. To date, only six proteins have been found necessary for the second step of splicing in yeast, and their human homologs have been identified. We demonstrate that the addition of the selective chelator of zinc, 1,10-phenanthroline, to an in vitro mRNA splicing reaction causes a dose-dependent inhibition of the second step of splicing. This inhibition is accompanied by the accumulation of spliceosomes paused before completion of step two of the splicing reaction. The inhibition effect on the second step is due neither to snRNA degradation nor to direct binding to the mRNA, and is reversible by dialysis or add-back of zinc, but not of other divalent metals, at the beginning of the reaction. These findings suggest that the activity of a putative zinc-dependent metalloprotein(s) involved in the second step of splicing is affected. This study outlines a new method for specific reversible inhibition of the second step of splicing using external reagents, and suggests a possible role of divalent cations in the second step of mRNA splicing, most likely zinc.

An artificial riboswitch for controlling pre-mRNA splicing

“…lanes 4-6 and lanes 10-12, 16-18, 22-24). Quantitation of these results presented in Figure 6B indicates that theophylline-dependent inhibition of the step II of splicing leads to the accumulation of complex B/C, which is consistent with previously published reports in which mutation of the 3 0 ss (Gozani et al 1994) or the addition of boric acid, both of which specifically inhibit step II of the splicing, led to the accumulation of complexes B/C (Shomron et al 2002;Shomron and Ast, 2003).…”

Molecular architecture of zinc chelating small molecules that inhibit spliceosome assembly at an early stage

“…mechanism of action of the compounds described. However, one should note the dramatic difference between the effects observed here, and those of the zinc chelation by 1,10-phe, which requires millimolar concentrations and results in the reversible inhibition of step 2 of splicing (Table 1; Shomron et al 2002).…”

“…Additionally, the activity of histone deacetylases (HDACs), which are classical zinc metalloproteases, has been shown to be important for splicing catalysis, although their precise functions in the splicing process are not clear (Hnilicová et al 2011). In fact, zinc depletion by the chelator 1,10-phenanthroline (1,10-phe) has been shown to inhibit the second step of splicing (Shomron et al 2002). It has been demonstrated that specific zinc-chelating agents that perturb the acetylation state of proteins (through inhibition of HDACs activity) block the splicing cycle (Kuhn et al 2009).…”

Splicing Factor hSlu7 Contains a Unique Functional Domain Required to Retain the Protein within the Nucleus

“…Combinatorial control of different nuclear protein concentrations varying among tissues (Hanamura et al, 1998) and cell types (Jensen et al, 2000) or during development (Mahe et al, 2000) was shown to determine exon choice (Wang and Manley, 1995;Smith and Valcarcel, 2000;Lallena et al, 2002), and these can be altered upon physiological stimuli (van der Houven van Oordt et al, 2000), environmental effects , or phosphorylation state (Xie et al, 2003). Aberrant regulation of splicing has been implicated in an increasing number of human diseases, including cancer (Hastings and Krainer, 2001;Modrek and Lee, 2002;Stoilov et al, 2002;Changela et al, 2003).In a previous study, we showed the possible role of zinc, or a metalloprotein, in the second step of splicing in vitro (Shomron et al, 2002;Shomron and Ast, 2003). Zinc is an essential element for all organisms as a catalytic and/or structural cofactor for many zinc-dependent enzymes and proteins.…”

“…In a previous study, we showed the possible role of zinc, or a metalloprotein, in the second step of splicing in vitro (Shomron et al, 2002;Shomron and Ast, 2003). Zinc is an essential element for all organisms as a catalytic and/or structural cofactor for many zinc-dependent enzymes and proteins.…”