TY - JOUR

T1 - RNAi-mediated depletion of the NSL complex subunits leads to abnormal chromosome segregation and defective centrosome duplication in Drosophila mitosis

AU - Pavlova, Gera A.

AU - Popova, Julia V.

AU - Andreyeva, Evgeniya N.

AU - Yarinich, Lyubov A.

AU - Lebedev, Mikhail O.

AU - Razuvaeva, Alyona V.

AU - Dubatolova, Tatiana D.

AU - Oshchepkova, Anastasiya L.

AU - Pellacani, Claudia

AU - Somma, Maria Patrizia

AU - Pindyurin, Alexey V.

AU - Gatti, Maurizio

PY - 2019/9/1

Y1 - 2019/9/1

N2 - The Drosophila Nonspecific Lethal (NSL) complex is a major transcriptional regulator of housekeeping genes. It contains at least seven subunits that are conserved in the human KANSL complex: Nsl1/Wah (KANSL1), Dgt1/Nsl2 (KANSL2), Rcd1/Nsl3 (KANSL3), Rcd5 (MCRS1), MBD-R2 (PHF20), Wds (WDR5) and Mof (MOF/KAT8). Previous studies have shown that Dgt1, Rcd1 and Rcd5 are implicated in centrosome maintenance. Here, we analyzed the mitotic phenotypes caused by RNAi-mediated depletion of Rcd1, Rcd5, MBD-R2 or Wds in greater detail. Depletion of any of these proteins in Drosophila S2 cells led to defects in chromosome segregation. Consistent with these findings, Rcd1, Rcd5 and MBD-R2 RNAi cells showed reduced levels of both Cid/CENP-A and the kinetochore component Ndc80. In addition, RNAi against any of the four genes negatively affected centriole duplication. In Wds-depleted cells, the mitotic phenotypes were similar but milder than those observed in Rcd1-, Rcd5- or MBD-R2-deficient cells. RT-qPCR experiments and interrogation of published datasets revealed that transcription of many genes encoding centromere/kinetochore proteins (e.g., cid, Mis12 and Nnf1b), or involved in centriole duplication (e.g., Sas-6, Sas-4 and asl) is substantially reduced in Rcd1, Rcd5 and MBD-R2 RNAi cells, and to a lesser extent in wds RNAi cells. During mitosis, both Rcd1-GFP and Rcd5-GFP accumulate at the centrosomes and the telophase midbody, MBD-R2-GFP is enriched only at the chromosomes, while Wds-GFP accumulates at the centrosomes, the kinetochores, the midbody, and on a specific chromosome region. Collectively, our results suggest that the mitotic phenotypes caused by Rcd1, Rcd5, MBD-R2 or Wds depletion are primarily due to reduced transcription of genes involved in kinetochore assembly and centriole duplication. The differences in the subcellular localizations of the NSL components may reflect direct mitotic functions that are difficult to detect at the phenotypic level, because they are masked by the transcription-dependent deficiency of kinetochore and centriolar proteins.Author summary The Drosophila Nonspecific Lethal (NSL) complex is a conserved protein assembly that controls transcription of more than 4,000 housekeeping genes. We analyzed the mitotic functions of four genes, Rcd1, Rcd5, MBD-R2 and wds, encoding NSL subunits. Inactivation of these genes by RNA interference (RNAi) resulted in defects in both chromosome segregation and centrosome duplication. Our analyses indicate that RNAi against Rcd1, Rcd5 or MBD-R2 reduces transcription of genes involved in centromere/kinetochore assembly and centriole replication. During interphase, Rcd1, Rcd5, MBD-R2 and Wds are confined to the nucleus, as expected for transcription factors. However, during mitosis each of these proteins relocates to specific mitotic structures. Our results suggest that the four NSL components work together as a complex to stimulate transcription of genes encoding important mitotic determinants. However, the different localization of the proteins during mitosis suggests that they might have acquired secondary "moonlighting" functions that directly contribute to the mitotic process.

AB - The Drosophila Nonspecific Lethal (NSL) complex is a major transcriptional regulator of housekeeping genes. It contains at least seven subunits that are conserved in the human KANSL complex: Nsl1/Wah (KANSL1), Dgt1/Nsl2 (KANSL2), Rcd1/Nsl3 (KANSL3), Rcd5 (MCRS1), MBD-R2 (PHF20), Wds (WDR5) and Mof (MOF/KAT8). Previous studies have shown that Dgt1, Rcd1 and Rcd5 are implicated in centrosome maintenance. Here, we analyzed the mitotic phenotypes caused by RNAi-mediated depletion of Rcd1, Rcd5, MBD-R2 or Wds in greater detail. Depletion of any of these proteins in Drosophila S2 cells led to defects in chromosome segregation. Consistent with these findings, Rcd1, Rcd5 and MBD-R2 RNAi cells showed reduced levels of both Cid/CENP-A and the kinetochore component Ndc80. In addition, RNAi against any of the four genes negatively affected centriole duplication. In Wds-depleted cells, the mitotic phenotypes were similar but milder than those observed in Rcd1-, Rcd5- or MBD-R2-deficient cells. RT-qPCR experiments and interrogation of published datasets revealed that transcription of many genes encoding centromere/kinetochore proteins (e.g., cid, Mis12 and Nnf1b), or involved in centriole duplication (e.g., Sas-6, Sas-4 and asl) is substantially reduced in Rcd1, Rcd5 and MBD-R2 RNAi cells, and to a lesser extent in wds RNAi cells. During mitosis, both Rcd1-GFP and Rcd5-GFP accumulate at the centrosomes and the telophase midbody, MBD-R2-GFP is enriched only at the chromosomes, while Wds-GFP accumulates at the centrosomes, the kinetochores, the midbody, and on a specific chromosome region. Collectively, our results suggest that the mitotic phenotypes caused by Rcd1, Rcd5, MBD-R2 or Wds depletion are primarily due to reduced transcription of genes involved in kinetochore assembly and centriole duplication. The differences in the subcellular localizations of the NSL components may reflect direct mitotic functions that are difficult to detect at the phenotypic level, because they are masked by the transcription-dependent deficiency of kinetochore and centriolar proteins.Author summary The Drosophila Nonspecific Lethal (NSL) complex is a conserved protein assembly that controls transcription of more than 4,000 housekeeping genes. We analyzed the mitotic functions of four genes, Rcd1, Rcd5, MBD-R2 and wds, encoding NSL subunits. Inactivation of these genes by RNA interference (RNAi) resulted in defects in both chromosome segregation and centrosome duplication. Our analyses indicate that RNAi against Rcd1, Rcd5 or MBD-R2 reduces transcription of genes involved in centromere/kinetochore assembly and centriole replication. During interphase, Rcd1, Rcd5, MBD-R2 and Wds are confined to the nucleus, as expected for transcription factors. However, during mitosis each of these proteins relocates to specific mitotic structures. Our results suggest that the four NSL components work together as a complex to stimulate transcription of genes encoding important mitotic determinants. However, the different localization of the proteins during mitosis suggests that they might have acquired secondary "moonlighting" functions that directly contribute to the mitotic process.

KW - CENTRIOLE DUPLICATION

KW - KANSL1 CAUSE

KW - CYTOKINESIS

KW - MUTATIONS

KW - ASTERLESS

KW - MIDBODY

KW - REVEALS

KW - ORGANIZATION

KW - DISSECTION

KW - COMPONENT

KW - Chromosome Duplication/genetics

KW - Regulatory Elements, Transcriptional/genetics

KW - Drosophila Proteins/genetics

KW - Centromere/metabolism

KW - RNA-Binding Proteins/genetics

KW - Protein Transport/physiology

KW - RNA Interference

KW - Nuclear Proteins/genetics

KW - Mitosis/genetics

KW - Vesicular Transport Proteins/genetics

KW - Kinetochores/metabolism

KW - Spindle Apparatus/genetics

KW - Chromosome Segregation/genetics

KW - Transcription Factors/genetics

KW - Microtubules/metabolism

KW - Drosophila melanogaster/genetics

KW - Animals

KW - Cell Cycle Proteins/genetics

KW - Centrosome/metabolism

UR - http://www.scopus.com/inward/record.url?scp=85072848578&partnerID=8YFLogxK

U2 - 10.1371/journal.pgen.1008371

DO - 10.1371/journal.pgen.1008371

M3 - Article

C2 - 31527906

AN - SCOPUS:85072848578

VL - 15

SP - e1008371

JO - PLoS Genetics

JF - PLoS Genetics

SN - 1553-7390

IS - 9

M1 - e1008371

ER -