Unknown,Transcriptomics,Genomics,Proteomics

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Experimental characterization of the human non-sequence-specific nucleic acid interactome

ABSTRACT: The interactions between proteins and nucleic acids have a fundamental function in many biological processes well beyond nuclear gene transcription and include RNA homeostasis, protein translation and pathogen sensing for innate immunity. While our knowledge of the ensemble of proteins binding individual mRNAs in mammalian cells has greatly been augmented by recent surveys, no systematic study on the native proteins of human cells differentially engaging various types of nucleic acids in a non sequence-specific manner has been reported. We designed an experimental approach to cover the non sequence-specific RNA and DNA binding space broadly, including methylation, and test for its ability to interact with the human proteome. We used 25 rationally designed nucleic acid probes in an affinity purification mass spectrometry and bioinformatics workflow to identify proteins from whole cell extracts of three different human cell lines. The proteins were profiled for their binding preferences to the different general types of nucleic acids. The study identified 746 high confidence direct binders, 249 of which were devoid of previous experimental evidence for binding nucleic acids. We could assign 513 specific affinities for sub-types of nucleic acid probes to 219 distinct proteins and to individual domains. The evolutionary conserved protein YB-1, previously associated with cancer and gene regulation, is shown to bind methylated cytosine preferentially conferring YB-1 a potential epigenetic function. Collectively, the dataset represents a rich resource of experimentally determined nucleic acid-specific binding proteins in humans and, indirectly, for other species. Identification of genomic YB-1 binding sites in HEK293 cells

ORGANISM(S): Homo sapiens

SUBMITTER: Gerhard Duernberger

PROVIDER: E-GEOD-47539 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Systemic lupus erythematosus is a chronic autoimmune disease with multifactorial ethiopathogenesis. The complement system is involved in both the early and late stages of disease development and organ damage. To better understand autoantibody mediated complement consumption the GAPAID consortium examined ex vivo immune complex formation on autoantigen arrays. We recruited patients with SLE (n=211), with other systemic autoimmune diseases (n=65) and non-autoimmune control subjects (n=149) in two rheumatology tertiary care centers. Standard clinical and laboratory data were collected from all subjects and serum complement levels were determined in SLE patients. The genotype of SNP rs1143679 in the ITGAM gene was also determined. On-chip formation of immune complexes was examined using a functional immunoassay on autoantigen microarray. The amount of antigen-bound IgM, IgG and complement C4 and C3 was quantified on autoantigens comprising nucleic acids, proteins and lipids. Our results show that the relatively high complement consumption of nucleic acids is further increased upon binding of IgM and IgG. This is true even when serum complement levels are decreased due to complement consumption in SLE patients. A negative correlation between serum complement levels and ex vivo complement deposition on nucleic acid autoantigens is demonstrated. On the contrary, most protein and lipid autoantigens show positive correlation with C4 and C3 levels. Genetic analysis reveals that the non-synonymous variant rs1143679 in complement receptor type 3 is associated with an increased production of anti-dsDNA IgG antibodies. Notwithstanding, homozygous carriers of the previously reported susceptible allele (AA) have lower levels of dsDNA specific IgM among SLE patients. Regarding organ involvement we find that besides anti-C1q IgG, low levels of dsDNA specific IgM and low complement C4 binding to C1q are also associated with renal injury. In summary, nucleic acids maintain a skewed complement deposition balance when bound by IgG and IgM, depleting the early classical complement pathway from other physiological processes. Dysfunction of the receptor responsible for complement-mediated apoptotic debris removal promotes the development of autoantibodies targeting nucleic acids. These observations provide serological and genetic evidence for complement-mediated clearance deficiency of apoptotic debris in lupus.

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Experimental characterization of the human non-sequence-specific nucleic acid interactome

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