Grad-seq

Grad-seq profiles gradient-separated cellular RNAs and proteins to resolve RNA–protein complexes and multisubunit protein assemblies in photosynthetic cyanobacteria such as Synechocystis 6803.

Key Features:

• High-resolution profiling: Distinguishes complexes with overlapping subunits, including CpcG1-type versus CpcL-type phycobilisomes and PsaK1 versus PsaK2 photosystem I pre-complexes.
• Identification of RNA chaperones: Uses clustering of in-gradient distribution profiles and additional selection criteria to nominate candidate RNA chaperones such as a YlxR homolog and a cyanobacterial KhpA/B homolog.
• Discovery of novel complexes: Reveals previously undetected interactions, including associations between accessory proteins and CRISPR-Cas systems and the Csx1-Csm6 ribonucleolytic defense complex.
• Exclusive association detection: Identifies exclusive co-migration of components such as RpoZ or 6S RNA with the core RNA polymerase complex.
• Sigma–antisigma complex reservoir: Detects evidence for inactive sigma–antisigma complexes relevant to transcriptional regulation.

Scientific Applications:

• Functional assignment of complexes: Enables annotation and subunit-level discrimination of RNA–protein and multisubunit protein complexes in photosynthetic organisms.
• sRNA regulatory network analysis: Facilitates exploration of small RNA (sRNA) interactions and their regulatory mechanisms.
• RNA chaperone characterization: Supports discovery and prioritization of candidate RNA chaperones for experimental validation.
• CRISPR-Cas interaction studies: Allows investigation of accessory protein associations and ribonucleolytic defense complexes such as Csx1-Csm6.
• Transcriptional regulation studies: Permits analysis of RNA polymerase composition and sigma–antisigma dynamics affecting transcription regulation.

Methodology:

Gradient separation of cellular fractions followed by sequencing to profile the full ensemble of RNAs and proteins, together with clustering of in-gradient distribution profiles and application of additional selection criteria.