Spliceosomal Prp8 intein at the crossroads of protein and RNA splicing

Abstract

protein splicing 15 16 * Corresponding authors: Marlene Belfort (mbelfort@albany.edu) or Hongmin Li 17 (hongmin.li@health.ny.gov) 18 ABSTRACT 19The spliceosome is a large ribonucleoprotein complex that removes introns from 20 pre-mRNAs. At its functional core lies the essential Prp8 protein. Across diverse 21 eukaryotes, this protein cofactor of RNA catalysis harbors a self-splicing element, called 22 an intein. Inteins in Prp8 are extremely pervasive and are found at seven different sites in 23 various species. Here, we focus on the Prp8 intein from Cryptococcus neoformans, a 24 human fungal pathogen. We solved the crystal structure of this intein, revealing structural 25 homology among self-splicing sequences in eukaryotes, including Hedgehog protein. 26Working with the C. neoformans Prp8 intein in a reporter assay, we find that the 27 biologically relevant divalent metals copper and zinc inhibit intein splicing, albeit by two 28 different mechanisms. Copper likely stimulates reversible modifications on a catalytically 29 important cysteine, whereas zinc binds via the same critical cysteine with a Kd of ~1 nM. 30An intein-containing Prp8 precursor model is presented, suggesting that metal-induced 31 protein splicing inhibition would disturb function of both Prp8 and the spliceosome. 32These results indicate that Prp8 protein splicing can be modulated, and that this could 33 alter spliceosome function and RNA splicing under specific conditions. 34 nuclear and chloroplast genomes with distinct patterns of insertion (27). Nuclear inteins 58 tend to be in proteins that are involved in energy metabolism and RNA processing, 59whereas chloroplast inteins are found in proteins that carry out transcription and 60replication. Out of all the intein-harboring proteins in eukaryotes, Prp8 is 61 overwhelmingly favored. There are over one hundred inteins identified across various 62 sites of Prp8 in different species. 63Pathogenic fungi seem to be enriched for inteins (27, 28). Several notable human 64 pathogens contain Prp8 inteins, including Aspergillus fumigatus, Histoplasma 65 capsulatum, and Cryptococcus neoformans. Intriguingly, many organisms with Prp8 66inteins also tend to be intron-rich (29). The presence of inteins in Prp8 and the correlation 67 with intron density beg the question of an intein benefit to the host, and especially to 68 pathogens. To begin to answer this question, we focus on the Prp8 intein from C. 69 neoformans. This is a mini-intein, naturally lacking the homing endonuclease domain, at 70 only 171 amino acid residues. The intein is also found at a highly conserved site at the 71 center of Prp8, and thus is at the core of the spliceosome (1, 5, 30). 72 Studying the Prp8 intein present in C. neoformans addresses questions of 73 conditional protein splicing in an important human pathogen in an entirely new domain 74 of life. Solving the Prp8 intein structure set the stage for beginning such studies and 75 provided evolutionary context, by revealing similarities to the metazoan Hedgehog 76 protein. B...

Conditional DnaB Protein Splicing Is Reversibly Inhibited by Zinc in Mycobacteria

“…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 .…”