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Principal Investigator: CHRISTOPH PROSCHEL
Organization: UNIVERSITY OF ROCHESTER
Fiscal Year: 2024
Award: $627,436
Funding agency: National Institute of Neurological Disorders and Stroke
PROJECT SUMMARY
The Integrator Complex (INT) is a 17-subunit machinery that associates with RNA polymerase II
(RNAPII) and functions as a critical transcription regulator. It is essential for the 3'-end formation of a
variety of non-coding RNAs and is a broad negative regulator of promoter-proximally paused RNAPII.
Integrator subunit 11 (INTS11) houses the RNA endonuclease domain vital for Integrator to cleave
nascent transcripts at all RNAPII loci, which is an important activity for transcriptional repression.
Consistent with a fundamental role in Integrator function, INTS11 genetic variants are found to disrupt
human development and give rise to a complex neurological syndrome. Similarly, genetic disruptions in
two non-Integrator proteins that associate with INTS11 also impede neurodevelopment, including
BRAT1, where variants cause a lethal neonatal multi-focus seizure syndrome and cerebellar ataxia, and
WDR73, where variants cause Galloway-Mowat syndrome that is characterized by psychomotor
impairment, hypotonia, and seizures.
WDR73 and BRAT1 associate with INTS11 and INTS9 but are primarily cytoplasmic proteins
distinct from the complete 17-subunit nuclear Integrator Complex. Notably, nothing is known about how
these proteins bind INTS11 or their role in nuclear Integrator function – especially in the context of
neuronal cell fate and fitness. Using biochemical and structural approaches, we generated cryo-EM
structures of the INTS11-BRAT1 binary complex and the INTS9-INTS11-BRAT1 ternary complex.
Surprisingly, we also find that optimal INTS11 function within the nuclear Integrator Complex requires
interaction with these cytoplasmic proteins as cells lacking BRAT1 or WDR73 accumulate uncleaved
Integrator substrates. Altogether, these preliminary studies generate a provocative model whereby
cytoplasmic BRAT1 and WDR73 are required to ‘license’ INTS11 for its nuclear activity, which is critical
to maintaining proper neuronal function in humans. To test this hypothesis, we propose these Specific
Aims: Specific Aim 1. Structurally and biochemically characterize the INTS9, INTS11, BRAT1, and
WDR73 complexes. Specific Aim 2. Probe the function of BRAT1 and WDR73 in Integrator-mediated
gene regulation. Specific Aim 3. Decipher the role of the INTS11-containing complexes in neural cells.
Terms: <Active Sites><Address><Architecture><Auxins><Binding><Binding Proteins><Biochemical><Biochemistry><Biological Chemistry><Brain><Brain Diseases><Brain Disorders><Brain Nervous System><C-terminal><CRISPR><CRISPR/Cas system><Cell Body><Cell Line><CellLine><Cells><Cerebellar Ataxia><Cerebellar Incoordination><Clustered Regularly Interspaced Short Palindromic Repeats><Complex><Cryo-electron Microscopy><Cryoelectron Microscopy><Cytoplasm><Cytoplasmic Protein><Cytoplasmic Structures><DNA-Dependent RNA Polymerase II><Decreased Muscle Tone><Defect><Electron Cryomicroscopy><Encephalon><Encephalon Diseases><Endoribonucleases><Engineering / Architecture><Functional RNA><Galloway syndrome><Galloway-Mowat syndrome><Gene Action Regulation><Gene Down-Regulation><Gene Expression><Gene Expression Regulation><Gene Regulation><Gene Regulation Process><Gene Transcription><Gene variant><Genes><Genetic><Genetic Alteration><Genetic Change><Genetic Transcription><Genetic defect><Glia><Glial Cells><Health><Human><Human Development><Human Engineering><Hypomyotonia><Hypotonia><Intracranial CNS Disorders><Intracranial Central Nervous System Disorders><Knowledge><Kolliker's reticulum><Laboratories><Licensing><Life><Ligand Binding Protein><Ligand Binding Protein Gene><Measures><Mediating><Modeling><Modern Man><Molecular><Molecular Interaction><Muscle Hypotony><Muscle Tone Poor><Muscle hypotonia><Muscular Hypotonia><Mutate><Mutation><Neonatal><Nerve Cells><Nerve Unit><Nervous System Diseases><Nervous System Disorder><Neural Cell><Neural Development><Neurobiology><Neurocyte><Neuroglia><Neuroglial Cells><Neurologic><Neurologic Disorders><Neurologic Manifestations><Neurologic Signs and Symptoms><Neurologic Symptoms><Neurological><Neurological Disorders><Neurological Manifestations><Neurological Signs and Symptoms><Neuronal Dysfunction><Neurons><Non-Coding><Non-Coding RNA><Non-neuronal cell><Non-translated RNA><Noncoding RNA><Nonneuronal cell><Nontranslated RNA><Nuclear><Null Cells><Null Lymphocytes><Organoids><Pathogenicity><Patients><Pattern><Process><Progenitor Cells><Property><Protein Binding><Proteins><Psychomotor Impairments><RNA Expression><RNA Polymerase B><RNA Polymerase II><RNA Seq><RNA endonuclease><RNA sequencing><RNAseq><Research><Resolution><Role><Seizures><Strains Cell Lines><Structure><Syndrome><System><Testing><Transcript><Transcription><Transcription Regulation><Transcription Repression><Transcriptional Control><Transcriptional Regulation><Transcriptional Repression><Untranslated RNA><Variant><Variation><allele variant><allelic variant><bound protein><brain cell><cell type><congenital microcephaly-hiatus hernia-nephrotic syndrome><cryo-EM><cryoEM><cryogenic electron microscopy><cultured cell line><disability><experiment><experimental research><experimental study><experiments><fitness><functional genomics><gene repression><genetic variant><genome editing><genome mutation><genomic editing><genomic variant><hESC><human ES cell><human ESC><human embryonic stem cell><microcephaly-hiatus hernia-nephrotic syndrome><microcephaly-infantile spasms-psychomotor retardation-nephrotic syndrome><mutant><nephrosis-microcephaly-hiatus hernia syndrome><nephrosis-neural dysmigration syndrome><nerve cement><neural dysfunction><neural manifestation><neurobiological><neurodevelopment><neurological disease><neuronal><noncoding><novel><promoter><promotor><resolutions><scRNA-seq><single cell RNA-seq><single cell RNAseq><single cell expression profiling><single cell transcriptomic profiling><single-cell RNA sequencing><small molecular inhibitor><small molecule inhibitor><social role><stem cells><structural biology><synergism><transcriptome sequencing><transcriptomic sequencing>