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A coilin mutant provides a sensitized system for identifying modifiers of alternative splicing and posttranscriptional gene silencing in Arabidopsis thaliana

ABSTRACT: Coilin is a scaffold protein essential for the structural integrity of Cajal Bodies, which are non-membranous nuclear organelles that are thought to facilitate assembly and maturation of nuclear RNPs, including spliceosomal snRNPs. To investigate further coilin’s functions in plant cells, and to identify proteins that may functionally interact with coilin, we performed a genetic suppressor screen in Arabidopsis thaliana using a coilin (coi) mutant displaying altered splicing of a GFP pre-mRNA. The modified splicing pattern results in a ‘hyper-GFP’ phenotype in young coi seedlings relative to the intermediate level of GFP in wild-type seedlings. Additionally, in newly emerging leaves of older coi seedlings, the GFP gene frequently undergoes abrupt siRNA-associated posttranscriptional gene silencing that persists during growth. In the suppressor screen, we searched for mutations that subdue one or both of these GFP phenotypes and identified several understudied factors in plants: WRAP53, a putative Cajal body protein; SMU2, a predicted splicing-related factor; and ZC3HC1, an uncharacterized zinc finger protein. All three mutations return the hyper-GFP phenotype of the coi mutant to approximately the intermediate wild-type level. The zc3hc1 mutations in particular induce premature and more extensive posttranscriptional gene silencing similar to mutations in SOP1 and DCL4, which are known modifiers of posttranscriptional gene silencing. Candidate coilin-interacting proteins identified by immunoprecipitation-mass spectrometry include many splicing-related factors, nucleolar proteins, and mRNA export factors. Our results demonstrate the usefulness of the coi mutant to identify new modifiers of alternative splicing and posttranscriptional gene silencing, and suggest diverse roles for coilin in plant cells.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Arabidopsis Thaliana (mouse-ear Cress)

TISSUE(S): Whole Body

SUBMITTER: Chuan-Chih Hsu

LAB HEAD: Marjori Matzke

PROVIDER: PXD027146 | Pride | 2023-07-27

REPOSITORIES: pride

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			Action	DRS
	108097_Coilin_Flag_IP_R327_20190516_1-_1_.msf	Msf
	108097_Coilin_Flag_IP_R327_20190516_1.mzML	Mzml
	108097_Coilin_Flag_IP_R327_20190516_1.raw	Raw
	108151_Coilinm_RED_IP_R327_20190611_1-_1_.msf	Msf
	108151_Coilinm_RED_IP_R327_20190611_1.mzML	Mzml

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A coilin mutant provides a sensitized system for identifying modifiers of alternative splicing and posttranscriptional gene silencing in Arabidopsis thaliana

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