Summary Cleavage and polyadenylation factor (CPF/CPSF) is a multi-protein complex essential for formation of eukaryotic mRNA 3ʹ ends. CPF cleaves pre-mRNAs at a specific site and adds a poly(A) tail. The cleavage reaction defines the 3ʹ end of the mature mRNA, and thus the activity of the endonuclease is highly regulated. Here, we show that reconstitution of specific pre-mRNA cleavage with recombinant yeast proteins requires incorporation of the Ysh1 endonuclease into an eight-subunit “CPF core ” complex. Cleavage also requires the accessory cleavage factors IA and IB, which bind substrate pre-mRNAs and CPF, likely facilitating assembly of an active complex. Using X-ray crystallography, electron microscopy, and mass spectrometry, we determine the structure of Ysh1 bound to Mpe1 and the arrangement of subunits within CPF core . Together, our data suggest that the active mRNA 3ʹ end processing machinery is a dynamic assembly that is licensed to cleave only when all protein factors come together at the polyadenylation site.

Cut7, the sole kinesin-5 in Schizosaccharomyces pombe, is essential for mitosis. Like other yeast kinesin-5 motors, Cut7 can reverse its stepping direction, by mechanisms that are currently unclear. Here we show that for full-length Cut7, the key determinant of stepping direction is the degree of motor crowding on the microtubule lattice, with greater crowding converting the motor from minus enddirected to plus end-directed stepping. To explain how high Cut7 occupancy causes this reversal, we postulate a simple proximity sensing mechanism that operates via steric blocking. We propose that the minus end-directed stepping action of Cut7 is selectively inhibited by collisions with neighbors under crowded conditions, whereas its plus end-directed action, being less space-hungry, is not. In support of this idea, we show that the direction of Cut7-driven microtubule sliding can be reversed by crowding it with non-Cut7 proteins. Thus, crowding by either dynein microtubule binding domain or Klp2, a kinesin-14, converts Cut7 from net minus end-directed to net plus end-directed stepping. Biochemical assays confirm that the Cut7 N terminus increases Cut7 occupancy by binding directly to microtubules. Direct observation by cryoEM reveals that this occupancyenhancing N-terminal domain is partially ordered. Overall, our data point to a steric blocking mechanism for directional reversal through which collisions of Cut7 motor domains with their neighbors inhibit their minus end-directed stepping action, but not their plus enddirected stepping action. Our model can potentially reconcile a number of previous, apparently conflicting, observations and proposals for the reversal mechanism of yeast kinesins-5.Cut7 | kinesin-5 | bidirectional kinesin | mitotic kinesin | kinesin crowding K inesin-5 is essential for mitosis in many eukaryotes, from yeast to humans. Its main function appears to be to establish spindle bipolarity by driving the spindle poles apart during mitotic prophase (1). All kinesins-5 studied to date are homotetramers that crosslink and slide microtubules (MTs), and until recently, all were thought to be plus end-directed (2-8), meaning that their stepping action drives antiparallel MTs to slide slowly apart, with minus ends out. However, it was recently shown that a yeast kinesin-5, Saccharomyces cerevisae Cin8, not only can step toward MT plus ends, but also can move rapidly and processively toward MT minus ends (9, 10). The mechanism and biological significance of this bidirectionality are unknown.Several factors have already been shown to influence kinesin-5 directional switching. Cin8 is a tetramer (11) that steps toward plus ends when linking antiparallel MTs but toward minus ends when moving as a single molecule on un-crosslinked MTs. Crosslinking of two MTs is required for plus end-directed stepping (5), such that the engagement of only one MT turns off plus end-directed motility but supports minus end-directed motility (10, 12). The MT sliding direction of Cin8 also has been shown to reverse according to th...