Frédéric Halgand

Proteomic analysis identifies a new complex required for nuclear pre‐mRNA retention and splicing

Using the proteomic tandem affinity purification (TAP) method, we have purified the Saccharomyces cerevisie U2 snRNP‐associated splicing factors SF3a and SF3b. While SF3a purification revealed only the expected subunits Prp9p, Prp11p and Prp21p, yeast SF3b was found to contain only six subunits, including previously known components (Rse1p, Hsh155p, Cus1p, Hsh49p), the recently identified Rds3p factor and a new small essential protein (Ysf3p) encoded by an unpredicted split ORF in the yeast genome. Surprisingly, Snu17p, the proposed yeast orthologue of the seventh human SF3b subunit, p14, was not found in the yeast complex. TAP purification revealed that Snu17p, together with Bud13p and a newly identified factor, Pml1p/Ylr016c, form a novel trimeric complex. Subunits of this complex were not essential for viability. However, they are required for efficient splicing in vitro and in vivo. Furthermore, inactivation of this complex causes pre‐mRNA leakage from the nucleus. The corresponding complex was named pre‐mRNA REtention and Splicing (RES). The presence of RES subunit homologues in numerous eukaryotes suggests that its function is evolutionarily conserved.

Stability study of Rhodobacter capsulatus ferrocytochrome c2 wild-type and site-directed mutants using hydrogen/deuterium exchange monitored by electrospray ionization mass spectrometry☆

To estimate the stability of Rhodobacter capsulatus ferrocytochrome c 2 wild‐type and site‐directed mutants, charge state distributions and hydrogen/deuterium exchange rates were monitored by electrospray ionization mass spectrometry. The relative stability of the mutants was observed with the order: V11 insert > Y75F > wild‐type = K32E > K12D = K14E ≥ K52E > K14E/K32E > W67Y > P35A > 157N > G34S. (A preliminary account has been presented for mutants G34S and P35A [Jaquinod et al. (1995) Rapid Commun. Mass Spectrom. 9, 1135–1140].) This approach is shown to be a useful tool for rapid characterization of mutational effects on protein conformation.