Legends for Supplementary Figures

Fig. S1. Phylogenetic analysis of RNase III proteins

Amino acid sequences of the conserved part of the second RNase III domains of Drosha and Dicer-like proteins were used to construct a phylogenetic tree using the neighbor- joining method. The homologous part of the RNase III domain of Pac1 (an E. coli RNase

III-like protein) was used as an out group. Accession numbers of proteins and their amino acid residues used for the analysis are follows: Pac1, AAU05315, 141aa-280aa; Drosha

(Hs), NP_037367, 1123-1252; Drosha (Dm), AAD31170, 734-860; Drosha (Ce),

NP_492599, 849-975; Dcr2p, BAD34723, 1598-1756; Dcr1p, BAD34722, 1169-1360;

Dcl1p, BAD34724, 1027-1181; DCL1, Q9SP32, 1575-1727; DCR-1, NP_683750, 1666-

1829; Dicer-1, Q9VCU9, 2009-2170; Dcr1, CAB37423, 1099-1253. At, Arabidopsis thaliana; Ce, Caenorhabditis elegans; Dm, Drosophila melanogaster; Hs, Homo sapiens;

Mm, Mus musculus; Sp, Schizosaccharomyces pombe; Tt, Tetrahymena thermophila.

Figure S2. Construction of the DCL1-HA strain

(A) Schematic drawings of the wild-type DCL1 locus and the HA tagged DCL1 (DCL1-

HA) locus. The HA coding sequence was inserted just before the translation termination codon (*) of DCL1. For selection, the neo3 cassette was introduced into the NsiI site (N) in the 3’-flanking region. A, AccI; Met, initiator methionine codon. (B) Confirmation of complete replacement of endogenous DCL1 loci by the DCL1-HA constructs. Total

DNAs isolated from wild-type CU428 (lane W) and DCL1-HA strain (lane H) were digested with AccI and the Southern blot was hybridized with the probe shown in (A). Figure S3. DCL1 knockout cells have defect in micronuclear chromosomes segregation and are hypersensitive to a microtubule de-polymerizing drug

(A) Cells containing anaphase bridges, lagging or fragment of micronuclear chromosomes at anaphase and telophase of mitosis were counted in a DCL1 knockout RI strain (DCL1-14A-RI ) and in wild-type strains (B2086 and CU428). (B, C) DCL1 knockout RI strains (DCL1-10-RI and DCL1-14A-RI) and a wild-type strain (B2086) were grown in medium without (B) or with (C) 10 M Oryzalin, a microtubule destabilizing drug. Growth was monitored by counting cells using a model ZB1 Coulter counter (Coulter Electronics Inc., Hialeah, FL)