RESUMEN
The ClpC1:ClpP1P2 protease is a core component of the proteostasis system in mycobacteria. To improve the efficacy of antitubercular agents targeting the Clp protease, we characterized the mechanism of the antibiotics cyclomarin A and ecumicin. Quantitative proteomics revealed that the antibiotics cause massive proteome imbalances, including upregulation of two unannotated yet conserved stress response factors, ClpC2 and ClpC3. These proteins likely protect the Clp protease from excessive amounts of misfolded proteins or from cyclomarin A, which we show to mimic damaged proteins. To overcome the Clp security system, we developed a BacPROTAC that induces degradation of ClpC1 together with its ClpC2 caretaker. The dual Clp degrader, built from linked cyclomarin A heads, was highly efficient in killing pathogenic Mycobacterium tuberculosis, with >100-fold increased potency over the parent antibiotic. Together, our data reveal Clp scavenger proteins as important proteostasis safeguards and highlight the potential of BacPROTACs as future antibiotics.
Asunto(s)
Antituberculosos , Mycobacterium tuberculosis , Antituberculosos/farmacología , Proteínas Bacterianas/metabolismo , Endopeptidasa Clp/metabolismo , Proteínas de Choque Térmico/metabolismo , Mycobacterium tuberculosis/efectos de los fármacos , ProteostasisRESUMEN
Methods to direct the degradation of protein targets with proximity-inducing molecules that coopt the cellular degradation machinery are advancing in leaps and bounds, and diverse modalities are emerging. The most used and well-studied approach is to hijack E3 ligases of the ubiquitin-proteasome system. E3 ligases use specific molecular recognition to determine which proteins in the cell are ubiquitinated and degraded. This review focuses on the structural determinants of E3 ligase recruitment of natural substrates and neo-substrates obtained through monovalent molecular glues and bivalent proteolysis-targeting chimeras. We use structures to illustrate the different types of substrate recognition and assess the basis for neo-protein-protein interactions in ternary complex structures. The emerging structural and mechanistic complexity is reflective of the diverse physiological roles of protein ubiquitination. This molecular insight is also guiding the application of structure-based design approaches to the development of new and existing degraders as chemical tools and therapeutics.
Asunto(s)
Ubiquitina-Proteína Ligasas , Ubiquitina , Proteínas/metabolismo , Proteolisis , Especificidad por Sustrato , Ubiquitina/genética , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , UbiquitinaciónRESUMEN
Hijacking the cellular protein degradation system offers unique opportunities for drug discovery, as exemplified by proteolysis-targeting chimeras. Despite their great promise for medical chemistry, so far, it has not been possible to reprogram the bacterial degradation machinery to interfere with microbial infections. Here, we develop small-molecule degraders, so-called BacPROTACs, that bind to the substrate receptor of the ClpC:ClpP protease, priming neo-substrates for degradation. In addition to their targeting function, BacPROTACs activate ClpC, transforming the resting unfoldase into its functional state. The induced higher-order oligomer was visualized by cryo-EM analysis, providing a structural snapshot of activated ClpC unfolding a protein substrate. Finally, drug susceptibility and degradation assays performed in mycobacteria demonstrate in vivo activity of BacPROTACs, allowing selective targeting of endogenous proteins via fusion to an established degron. In addition to guiding antibiotic discovery, the BacPROTAC technology presents a versatile research tool enabling the inducible degradation of bacterial proteins.
Asunto(s)
Proteínas Bacterianas , Chaperonas Moleculares , Bacterias/metabolismo , Proteínas Bacterianas/metabolismo , Chaperonas Moleculares/metabolismo , ProteolisisRESUMEN
Certain obligate parasites induce complex and substantial phenotypic changes in their hosts in ways that favor their transmission to other trophic levels. However, the mechanisms underlying these changes remain largely unknown. Here we demonstrate how SAP05 protein effectors from insect-vectored plant pathogenic phytoplasmas take control of several plant developmental processes. These effectors simultaneously prolong the host lifespan and induce witches' broom-like proliferations of leaf and sterile shoots, organs colonized by phytoplasmas and vectors. SAP05 acts by mediating the concurrent degradation of SPL and GATA developmental regulators via a process that relies on hijacking the plant ubiquitin receptor RPN10 independent of substrate ubiquitination. RPN10 is highly conserved among eukaryotes, but SAP05 does not bind insect vector RPN10. A two-amino-acid substitution within plant RPN10 generates a functional variant that is resistant to SAP05 activities. Therefore, one effector protein enables obligate parasitic phytoplasmas to induce a plethora of developmental phenotypes in their hosts.
Asunto(s)
Arabidopsis/crecimiento & desarrollo , Arabidopsis/parasitología , Interacciones Huésped-Parásitos/fisiología , Parásitos/fisiología , Proteolisis , Ubiquitinas/metabolismo , Secuencia de Aminoácidos , Animales , Arabidopsis/genética , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Ingeniería Genética , Humanos , Insectos/fisiología , Modelos Biológicos , Fenotipo , Fotoperiodo , Filogenia , Phytoplasma/fisiología , Desarrollo de la Planta , Brotes de la Planta/crecimiento & desarrollo , Plantas Modificadas Genéticamente , Complejo de la Endopetidasa Proteasomal/metabolismo , Estabilidad Proteica , Reproducción , Nicotiana , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
While cellular proteins were initially thought to be stable, research over the last decades has firmly established that intracellular protein degradation is an active and highly regulated process: Lysosomal, proteasomal, and mitochondrial degradation systems were identified and found to be involved in a staggering number of biological functions. Here, we provide a global overview of the diverse roles of cellular protein degradation using seven categories: homeostasis, regulation, quality control, stoichiometry control, proteome remodeling, immune surveillance, and baseline turnover. Using selected examples, we outline how proteins are degraded and why this is functionally relevant.
Asunto(s)
Autofagia , Proteoma , Autofagia/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteolisis , Proteoma/metabolismo , UbiquitinaciónRESUMEN
Stalled protein synthesis produces defective nascent chains that can harm cells. In response, cells degrade these nascent chains via a process called ribosome-associated quality control (RQC). Here, we review the irregularities in the translation process that cause ribosomes to stall as well as how cells use RQC to detect stalled ribosomes, ubiquitylate their tethered nascent chains, and deliver the ubiquitylated nascent chains to the proteasome. We additionally summarize how cells respond to RQC failure.
Asunto(s)
Escherichia coli/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Biosíntesis de Proteínas , Procesamiento Proteico-Postraduccional , Ribosomas/genética , Escherichia coli/metabolismo , Humanos , Modelos Moleculares , Poli A/química , Poli A/genética , Poli A/metabolismo , Complejo de la Endopetidasa Proteasomal/genética , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Estructura Secundaria de Proteína , Proteolisis , Empalme del ARN , Estabilidad del ARN , Ribosomas/metabolismo , Ribosomas/ultraestructura , Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , UbiquitinaciónRESUMEN
Electrophilic compounds originating from nature or chemical synthesis have profound effects on immune cells. These compounds are thought to act by cysteine modification to alter the functions of immune-relevant proteins; however, our understanding of electrophile-sensitive cysteines in the human immune proteome remains limited. Here, we present a global map of cysteines in primary human T cells that are susceptible to covalent modification by electrophilic small molecules. More than 3,000 covalently liganded cysteines were found on functionally and structurally diverse proteins, including many that play fundamental roles in immunology. We further show that electrophilic compounds can impair T cell activation by distinct mechanisms involving the direct functional perturbation and/or degradation of proteins. Our findings reveal a rich content of ligandable cysteines in human T cells and point to electrophilic small molecules as a fertile source for chemical probes and ultimately therapeutics that modulate immunological processes and their associated disorders.
Asunto(s)
Cisteína/metabolismo , Ligandos , Linfocitos T/metabolismo , Acetamidas/química , Acetamidas/farmacología , Acrilamidas/química , Acrilamidas/farmacología , Células Cultivadas , Humanos , Proteínas Inhibidoras de la Apoptosis/metabolismo , Activación de Linfocitos/efectos de los fármacos , Proteínas Tirosina Quinasas/metabolismo , Proteolisis/efectos de los fármacos , Proteoma/química , Proteoma/metabolismo , Estereoisomerismo , Linfocitos T/citología , Linfocitos T/inmunología , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
The 26S proteasome is the principal macromolecular machine responsible for protein degradation in eukaryotes. However, little is known about the detailed kinetics and coordination of the underlying substrate-processing steps of the proteasome, and their correlation with observed conformational states. Here, we used reconstituted 26S proteasomes with unnatural amino-acid-attached fluorophores in a series of FRET- and anisotropy-based assays to probe substrate-proteasome interactions, the individual steps of the processing pathway, and the conformational state of the proteasome itself. We develop a complete kinetic picture of proteasomal degradation, which reveals that the engagement steps prior to substrate commitment are fast relative to subsequent deubiquitination, translocation, and unfolding. Furthermore, we find that non-ideal substrates are rapidly rejected by the proteasome, which thus employs a kinetic proofreading mechanism to ensure degradation fidelity and substrate prioritization.
Asunto(s)
Complejo de la Endopetidasa Proteasomal/metabolismo , Complejo de la Endopetidasa Proteasomal/fisiología , Anisotropía , Sitios de Unión/fisiología , Activación Enzimática , Cinética , Modelos Moleculares , Unión Proteica , Conformación Proteica , Procesamiento Proteico-Postraduccional/fisiología , Proteolisis , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Especificidad por Sustrato/fisiología , Ubiquitina/metabolismoRESUMEN
As the endpoint for the ubiquitin-proteasome system, the 26S proteasome is the principal proteolytic machine responsible for regulated protein degradation in eukaryotic cells. The proteasome's cellular functions range from general protein homeostasis and stress response to the control of vital processes such as cell division and signal transduction. To reliably process all the proteins presented to it in the complex cellular environment, the proteasome must combine high promiscuity with exceptional substrate selectivity. Recent structural and biochemical studies have shed new light on the many steps involved in proteasomal substrate processing, including recognition, deubiquitination, and ATP-driven translocation and unfolding. In addition, these studies revealed a complex conformational landscape that ensures proper substrate selection before the proteasome commits to processive degradation. These advances in our understanding of the proteasome's intricate machinery set the stage for future studies on how the proteasome functions as a major regulator of the eukaryotic proteome.
Asunto(s)
Complejo de la Endopetidasa Proteasomal/química , Complejo de la Endopetidasa Proteasomal/metabolismo , ATPasas Asociadas con Actividades Celulares Diversas/química , ATPasas Asociadas con Actividades Celulares Diversas/metabolismo , Enzimas Desubicuitinizantes/química , Enzimas Desubicuitinizantes/metabolismo , Humanos , Modelos Biológicos , Modelos Moleculares , Proteínas Motoras Moleculares/química , Proteínas Motoras Moleculares/metabolismo , Conformación Proteica , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Especificidad por Sustrato , Ubiquitina/química , Ubiquitina/metabolismoRESUMEN
Degrons are minimal elements that mediate the interaction of proteins with degradation machineries to promote proteolysis. Despite their central role in proteostasis, the number of known degrons remains small, and a facile technology to characterize them is lacking. Using a strategy combining global protein stability (GPS) profiling with a synthetic human peptidome, we identify thousands of peptides containing degron activity. Employing CRISPR screening, we establish that the stability of many proteins is regulated through degrons located at their C terminus. We characterize eight Cullin-RING E3 ubiquitin ligase (CRL) complex adaptors that regulate C-terminal degrons, including six CRL2 and two CRL4 complexes, and computationally implicate multiple non-CRLs in end recognition. Proteome analysis revealed that the C termini of eukaryotic proteins are depleted for C-terminal degrons, suggesting an E3-ligase-dependent modulation of proteome composition. Thus, we propose that a series of "C-end rules" operate to govern protein stability and shape the eukaryotic proteome.
Asunto(s)
Proteoma/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Secuencias de Aminoácidos , Animales , Antígenos de Neoplasias/metabolismo , Sistemas CRISPR-Cas/genética , Biología Computacional/métodos , Vectores Genéticos/genética , Vectores Genéticos/metabolismo , Células HEK293 , Humanos , Lentivirus/genética , Leupeptinas/farmacología , Sistemas de Lectura Abierta/genética , Péptidos/metabolismo , Complejo de la Endopetidasa Proteasomal/química , Complejo de la Endopetidasa Proteasomal/metabolismo , Estabilidad Proteica/efectos de los fármacos , Subunidades de Proteína/metabolismo , Proteolisis , Proteoma/genética , Receptores de Citocinas/genética , Receptores de Citocinas/metabolismoRESUMEN
This brief disquisition about the early history of studies on regulated protein degradation introduces several detailed reviews about the ubiquitin system and autophagy.
Asunto(s)
Complejo de la Endopetidasa Proteasomal/metabolismo , Complejos de Ubiquitina-Proteína Ligasa/metabolismo , Ubiquitina/metabolismo , Autofagia/genética , Células Eucariotas/citología , Células Eucariotas/metabolismo , Expresión Génica , Historia del Siglo XX , Historia del Siglo XXI , Humanos , Complejo de la Endopetidasa Proteasomal/genética , Complejo de la Endopetidasa Proteasomal/historia , Proteolisis , Ubiquitina/genética , Ubiquitina/historia , Complejos de Ubiquitina-Proteína Ligasa/genética , Complejos de Ubiquitina-Proteína Ligasa/historia , UbiquitinaciónRESUMEN
The ubiquitin proteasome pathway is responsible for most of the protein degradation in mammalian cells. Rates of degradation by this pathway have generally been assumed to be determined by rates of ubiquitylation. However, recent studies indicate that proteasome function is also tightly regulated and determines whether a ubiquitylated protein is destroyed or deubiquitylated and survives longer. This article reviews recent advances in our understanding of the proteasome's multistep ATP-dependent mechanism, its biochemical and structural features that ensure efficient proteolysis and ubiquitin recycling while preventing nonselective proteolysis, and the regulation of proteasome activity by interacting proteins and subunit modifications, especially phosphorylation.
Asunto(s)
Complejo de la Endopetidasa Proteasomal/química , Complejo de la Endopetidasa Proteasomal/metabolismo , Adenosina Trifosfatasas/metabolismo , Regulación Alostérica , Animales , Eucariontes/química , Eucariontes/metabolismo , Humanos , Fosforilación , Proteolisis , UbiquitinaciónRESUMEN
Methods for the targeted disruption of protein function have revolutionized science and greatly expedited the systematic characterization of genes. Two main approaches are currently used to disrupt protein function: DNA knockout and RNA interference, which act at the genome and mRNA level, respectively. A method that directly alters endogenous protein levels is currently not available. Here, we present Trim-Away, a technique to degrade endogenous proteins acutely in mammalian cells without prior modification of the genome or mRNA. Trim-Away harnesses the cellular protein degradation machinery to remove unmodified native proteins within minutes of application. This rapidity minimizes the risk that phenotypes are compensated and that secondary, non-specific defects accumulate over time. Because Trim-Away utilizes antibodies, it can be applied to a wide range of target proteins using off-the-shelf reagents. Trim-Away allows the study of protein function in diverse cell types, including non-dividing primary cells where genome- and RNA-targeting methods are limited.
Asunto(s)
Anticuerpos/química , Bioquímica/métodos , Transporte de Proteínas , Proteolisis , AnimalesRESUMEN
Most eukaryotic proteins are degraded by the 26S proteasome after modification with a polyubiquitin chain. Substrates lacking unstructured segments cannot be degraded directly and require prior unfolding by the Cdc48 ATPase (p97 or VCP in mammals) in complex with its ubiquitin-binding partner Ufd1-Npl4 (UN). Here, we use purified yeast components to reconstitute Cdc48-dependent degradation of well-folded model substrates by the proteasome. We show that a minimal system consists of the 26S proteasome, the Cdc48-UN ATPase complex, the proteasome cofactor Rad23, and the Cdc48 cofactors Ubx5 and Shp1. Rad23 and Ubx5 stimulate polyubiquitin binding to the 26S proteasome and the Cdc48-UN complex, respectively, allowing these machines to compete for substrates before and after their unfolding. Shp1 stimulates protein unfolding by the Cdc48-UN complex rather than substrate recruitment. Experiments in yeast cells confirm that many proteins undergo bidirectional substrate shuttling between the 26S proteasome and Cdc48 ATPase before being degraded.
Asunto(s)
Complejo de la Endopetidasa Proteasomal , Proteínas de Saccharomyces cerevisiae , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Poliubiquitina/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteolisis , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitina/metabolismo , Proteína que Contiene Valosina/genética , Proteína que Contiene Valosina/metabolismoRESUMEN
Myriad physiological and pathogenic processes are governed by protein levels and modifications. Controlled protein activity perturbation is essential to studying protein function in cells and animals. Based on Trim-Away technology, we screened for truncation variants of E3 ubiquitinase Trim21 with elevated efficiency (ΔTrim21) and developed multiple ΔTrim21-based targeted protein-degradation systems (ΔTrim-TPD) that can be transfected into host cells. Three ΔTrim-TPD variants are developed to enable chemical and light-triggered programmable activation of TPD in cells and animals. Specifically, we used ΔTrim-TPD for (1) red-light-triggered inhibition of HSV-1 virus proliferation by degrading the packaging protein gD, (2) for chemical-triggered control of the activity of Cas9/dCas9 protein for gene editing, and (3) for blue-light-triggered degradation of two tumor-associated proteins for spatiotemporal inhibition of melanoma tumor growth in mice. Our study demonstrates that multiple ΔTrim21-based controllable TPD systems provide powerful tools for basic biology research and highlight their potential biomedical applications.
Asunto(s)
Sistemas CRISPR-Cas , Edición Génica , Ratones , Animales , Proteína 9 Asociada a CRISPR/genética , Proteína 9 Asociada a CRISPR/metabolismo , Proteínas/metabolismo , Proteolisis , Mamíferos/metabolismoRESUMEN
The posttranslational modifier ubiquitin regulates most cellular processes. Its ability to form polymeric chains of distinct linkages is key to its diverse functionality. Yet, we still lack the experimental tools to induce linkage-specific polyubiquitylation of a protein of interest in cells. Here, we introduce a set of engineered ubiquitin protein ligases and matching ubiquitin acceptor tags for the rapid, inducible linear (M1-), K48-, or K63-linked polyubiquitylation of proteins in yeast and mammalian cells. By applying the so-called "Ubiquiton" system to proteasomal targeting and the endocytic pathway, we validate this tool for soluble cytoplasmic and nuclear as well as chromatin-associated and integral membrane proteins and demonstrate how it can be used to control the localization and stability of its targets. We expect that the Ubiquiton system will serve as a versatile, broadly applicable research tool to explore the signaling functions of polyubiquitin chains in many biological contexts.
Asunto(s)
Ubiquitina-Proteína Ligasas , Ubiquitina , Animales , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Poliubiquitina/genética , Poliubiquitina/metabolismo , Transducción de Señal , Complejo de la Endopetidasa Proteasomal/metabolismo , Ubiquitinación , Mamíferos/metabolismoRESUMEN
Cullin-RING ligases (CRLs) ubiquitylate specific substrates selected from other cellular proteins. Substrate discrimination and ubiquitin transferase activity were thought to be strictly separated. Substrates are recognized by substrate receptors, such as Fbox or BCbox proteins. Meanwhile, CRLs employ assorted ubiquitin-carrying enzymes (UCEs, which are a collection of E2 and ARIH-family E3s) specialized for either initial substrate ubiquitylation (priming) or forging poly-ubiquitin chains. We discovered specific human CRL-UCE pairings governing substrate priming. The results reveal pairing of CUL2-based CRLs and UBE2R-family UCEs in cells, essential for efficient PROTAC-induced neo-substrate degradation. Despite UBE2R2's intrinsic programming to catalyze poly-ubiquitylation, CUL2 employs this UCE for geometrically precise PROTAC-dependent ubiquitylation of a neo-substrate and for rapid priming of substrates recruited to diverse receptors. Cryo-EM structures illuminate how CUL2-based CRLs engage UBE2R2 to activate substrate ubiquitylation. Thus, pairing with a specific UCE overcomes E2 catalytic limitations to drive substrate ubiquitylation and targeted protein degradation.
Asunto(s)
Proteínas Cullin , Ubiquitina-Proteína Ligasas , Humanos , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas Cullin/genética , Proteínas Cullin/metabolismo , Ubiquitinación , Ubiquitina/metabolismo , Poliubiquitina/metabolismo , Proteínas Portadoras/metabolismoRESUMEN
Do young and old protein molecules have the same probability to be degraded? We addressed this question using metabolic pulse-chase labeling and quantitative mass spectrometry to obtain degradation profiles for thousands of proteins. We find that >10% of proteins are degraded non-exponentially. Specifically, proteins are less stable in the first few hours of their life and stabilize with age. Degradation profiles are conserved and similar in two cell types. Many non-exponentially degraded (NED) proteins are subunits of complexes that are produced in super-stoichiometric amounts relative to their exponentially degraded (ED) counterparts. Within complexes, NED proteins have larger interaction interfaces and assemble earlier than ED subunits. Amplifying genes encoding NED proteins increases their initial degradation. Consistently, decay profiles can predict protein level attenuation in aneuploid cells. Together, our data show that non-exponential degradation is common, conserved, and has important consequences for complex formation and regulation of protein abundance.
Asunto(s)
Estabilidad Proteica , Proteínas/metabolismo , Proteolisis , Alanina/análogos & derivados , Alanina/química , Aneuploidia , Línea Celular , Química Clic , Amplificación de Genes , Humanos , Cinética , Cadenas de Markov , Complejo de la Endopetidasa Proteasomal/química , Biosíntesis de Proteínas , Proteínas/química , Proteínas/genética , Proteoma , Ubiquitina/químicaRESUMEN
Various hormones, kinases, and stressors (fasting, heat shock) stimulate 26S proteasome activity. To understand how its capacity to degrade ubiquitylated proteins can increase, we studied mouse ZFAND5, which promotes protein degradation during muscle atrophy. Cryo-electron microscopy showed that ZFAND5 induces large conformational changes in the 19S regulatory particle. ZFAND5's AN1 Zn-finger domain interacts with the Rpt5 ATPase and its C terminus with Rpt1 ATPase and Rpn1, a ubiquitin-binding subunit. Upon proteasome binding, ZFAND5 widens the entrance of the substrate translocation channel, yet it associates only transiently with the proteasome. Dissociation of ZFAND5 then stimulates opening of the 20S proteasome gate. Using single-molecule microscopy, we showed that ZFAND5 binds ubiquitylated substrates, prolongs their association with proteasomes, and increases the likelihood that bound substrates undergo degradation, even though ZFAND5 dissociates before substrate deubiquitylation. These changes in proteasome conformation and reaction cycle can explain the accelerated degradation and suggest how other proteasome activators may stimulate proteolysis.
Asunto(s)
Complejo de la Endopetidasa Proteasomal , Animales , Ratones , Adenosina Trifosfatasas , Microscopía por Crioelectrón , CitoplasmaRESUMEN
RNA polymerase II (RNA Pol II) has been recognized as a passively regulated multi-subunit holoenzyme. However, the extent to which RNA Pol II subunits might be important beyond the RNA Pol II complex remains unclear. Here, fractions containing disassociated RPB3 (dRPB3) were identified by size exclusion chromatography in various cells. Through a unique strategy, i.e., "specific degradation of disassociated subunits (SDDS)," we demonstrated that dRPB3 functions as a regulatory component of RNA Pol II to enable the preferential control of 3' end processing of ribosomal protein genes directly through its N-terminal domain. Machine learning analysis of large-scale genomic features revealed that the little elongation complex (LEC) helps to specialize the functions of dRPB3. Mechanistically, dRPB3 facilitates CBC-PCF11 axis activity to increase the efficiency of 3' end processing. Furthermore, RPB3 is dynamically regulated during development and diseases. These findings suggest that RNA Pol II gains specific regulatory functions by trapping disassociated subunits in mammalian cells.