These observations suggest that myosin-II along with actin crosslinkers establish local cortical tension and elasticity, allowing for contractility independent of a circumferential cytoskeletal array. Furthermore, myosin-II and actin crosslinkers may influence each other as they modulate the dynamics and mechanics of cell-shape change.
Prp8 is the largest and most highly conserved protein in the spliceosome yet its mechanism of function is poorly understood. Our previous studies implicate Prp8 in control of spliceosome activation for the first catalytic step of splicing, because substitutions in five distinct regions (a-e) of Prp8 suppress a cold-sensitive block to activation caused by a mutation in U4 RNA. Catalytic activation of the spliceosome is thought to require unwinding of the U1 RNA͞5 splice site and U4͞U6 RNA helices by the Prp28 and Prp44͞Brr2 DExD͞H-box helicases, respectively. Here we show that mutations in regions a, d, and e of Prp8 exhibit allele-specific genetic interactions with mutations in Prp28, Prp44͞Brr2, and U6 RNA, respectively. These results indicate that Prp8 coordinates multiple processes in spliceosome activation and enable an initial correlation of Prp8 structure and function. Furthermore, additional genetic interactions with U4-cs1 support a two-state model for this RNA conformational switch and implicate another splicing factor, Prp31, in Prp8-mediated spliceosome activation.pre-mRNA splicing ͉ small nuclear RNA ͉ retinitis pigmentosa ͉ RNA helicases ͉ Prp31 T he removal of introns from nuclear pre-mRNA is catalyzed by a macromolecular complex called the spliceosome, which consists of five RNAs (U1, U2, U4, U5, and U6) and more than 70 proteins. Pre-mRNA splicing is thought to be RNA-catalyzed and involves two transesterification reactions (1). The accuracy and efficiency of splicing is achieved by means of a high degree of regulation, imposed by a complex series of protein-protein, protein-RNA, and RNA-RNA interactions. The spliceosome either assembles on the intron by way of stepwise recruitment of the constituent small nuclear ribonucleoproteins, each containing one RNA and several proteins (2), or a fully assembled spliceosome binds the intron as a unit (3). After the complete spliceosome has engaged an intron, it must undergo extensive conformational rearrangements to become catalytically active. Early steps in catalytic activation include unwinding of the U1 RNA͞5Ј splice site helix and unwinding of the U4͞U6 helices (Fig. 1). These unwinding events are thought to be catalyzed by two DExD͞H-box RNA helicases, Prp28 (4, 5) and Prp44͞Brr2 (6-8), respectively.We identified a cold-sensitive mutation in U4 RNA, called U4-cs1, which blocks catalytic activation at low temperature (9, 10). U4-cs1 has a triple nucleotide substitution that extends the U4͞U6 base-pairing, thereby masking the ''ACAGA'' sequence in U6 RNA that base pairs with the 5Ј splice site when U1 RNA is displaced (Fig. 1). We proposed that in the absence of proper U6 RNA͞5Ј splice site pairing, U4͞U6 RNA unwinding is inhibited. In support of this hypothesis, a cold-sensitive mutation in PRP44͞BRR2 (brr2-1) exacerbates (enhances) the U4-cs1 mutation (11).A selection for suppressors of the cold-sensitive growth defect of U4-cs1 yielded a mutation in the highly conserved 280-kDa splicing factor Prp8 (10). Prp8 is a U5 RNA-associated protein that con...
The ultimate goal of all signaling pathways in cytokinesis is to control the mechanical separation of the mother cell into two daughter cells. Because of the intrinsic mechanical nature of cytokinesis, it is essential to understand fully how cell shapes and the material properties of the cell are generated, how these shapes and material properties create force, and how motor proteins such as myosin-II modify the system to achieve successful cytokinesis. In this review (which is part of the Cytokinesis series), we discuss the relevant physical properties of cells, how these properties are measured and the basic models that are used to understand cell mechanics. Finally, we present our current understanding of how cytokinesis mechanics work.
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