Spliceosomal Prp8 intein at the crossroads of protein and RNA splicing

Abstract

The spliceosome is a large ribonucleoprotein complex that removes introns from pre-mRNAs. At its functional core lies the essential pre-mRNA processing factor 8 (Prp8) protein. Across diverse eukaryotes, this protein cofactor of RNA catalysis harbors a self-splicing element called an intein. Inteins in Prp8 are extremely pervasive and are found at 7 different sites in various species. Here, we focus on the Prp8 intein from Cryptococcus neoformans (Cne), a human fungal pathogen. We solved the crystal structure of this intein, revealing structural homology among protein splicing sequences in eukaryotes, including the Hedgehog C terminus. Working with the Cne Prp8 intein in a reporter assay, we find that the biologically relevant divalent metals copper and zinc inhibit intein splicing, albeit by 2 different mechanisms. Copper likely stimulates reversible modifications on a catalytically important cysteine, whereas zinc binds at the terminal asparagine and the same critical cysteine. Importantly, we also show that copper treatment inhibits Prp8 protein splicing in Cne. Lastly, an intein-containing Prp8 precursor model is presented, suggesting that metal-induced protein splicing inhibition would disturb function of both Prp8 and the spliceosome. These results indicate that Prp8 protein splicing can be modulated, with potential functional implications for the spliceosome.

Conditional DnaB Protein Splicing Is Reversibly Inhibited by Zinc in Mycobacteria

“…Zinc inhibits DnaBi1 splicing in the native mycobacterial host. Divalent cations have been shown to influence protein splicing, in some cases binding directly to the intein (21,34,35). Further, it has been shown that DnaBi1 is subject to conditional splicing regulation via the redox state of catalytic cysteines (7).…”

“…Inteins are often found in experimentally challenging organisms such as pathogenic bacteria (e.g., M. tuberculosis) (11,25), pathogenic fungi (e.g., Cryptococcus neoformans) (21,35), and extremophilic archaea (e.g., Pyrococcus horikoshii) (9, 10). As a result, studies examining conditional protein splicing (CPS) in the native host have proven difficult.…”

“…Divalent cations, in particular, zinc, platinum, and copper, have previously been shown to inhibit protein splicing of several inteins in vitro (34,35). Additionally, hedgehog autoprocessing in eukaryotes, which exhibites evolutionary, structural, and mechanistic similarities to protein splicing, is inhibited by zinc (41).…”

“…Zinc represents an intriguing candidate to block splicing. From the mechanistic perspective, zinc is not redox active, and thus cysteine oxidation is not required to block splicing, in contrast to copper (35). Presumably, reversible binding to the intein could pause splicing when zinc levels are high and could then be naturally titrated away as cellular levels decrease to allow splicing to proceed.…”

“…Many inteins are evolutionarily maintained in the active site of their natural host proteins (1), and interestingly, kanamycin resistance is dependent on splicing when the DnaBi1 is inserted near the KanR active site. Although no additional enzyme or cofactors are known to be required for splicing, metal ions have been found to inhibit splicing both in vivo and in vitro and to be bound to the active site of intein crystals (21,34,35). Using this reporter, we sought to examine whether zinc, a biologically relevant cation long known to inhibit protein splicing (34), blocks DnaBi1 splicing both in vivo and in vitro.…”