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Structure and Function in RNA Processing and Ubiquitin-like Protein Conjugation

Research Summary

Christopher Lima studies essential eukaryotic pathways that control co- and posttranscriptional RNA processing as well as posttranslational protein modification by ubiquitin and ubiquitin-like modifiers such as SUMO. He uses biophysical techniques, principally x-ray crystallography in conjunction with biochemistry and genetics, to elucidate mechanisms and to understand how these pathways contribute to signaling, cell cycle control, and regulation of RNA metabolism.

Posttranslational Protein Modification by Ubiquitin and Ubiquitin-like Modifiers
Ubiquitin (Ub) and ubiquitin-like (Ubl) protein conjugation pathways regulate nearly every facet of eukaryotic cell biology, including transcription, replication, chromosome segregation, the cell cycle, signaling, and DNA repair. Ub/Ubl conjugation requires a sequential cascade of activities that are catalyzed by evolutionarily related E1 activating enzymes, E2 conjugating enzymes, and E3 ligases that primarily belong to the RING and HECT-like protein families (Figure 1).

Substrate modification by Ub/Ubl proteins can promote protein-protein interactions, redistribution of subcellular localization, or alteration in the activities of the protein that is attached to the Ub/Ubl. Ub/Ubl pathways are often deregulated in human diseases such as cancer, in the immune response, and in neural degeneration, so understanding how these pathways function is a critical step toward understanding how pathway dysfunction contributes to disease. My lab has a long-standing interest in establishing biological functions for Ub and the Ubl protein SUMO by characterizing enzymes and factors involved in their conjugation and recognition.

E1s are gatekeepers for Ub/Ubl pathways because they are required to activate their Ub/Ubls and select a cognate E2 for transfer. My lab characterized several complexes in this cascade in studies that revealed that adenylation and thioester bond formation require large conformational changes to transit the E1 cysteine 35 Å into the active site to promote thioester bond transfer. The Cys domain then returns to its open state to engage the E2, and another conformational change is required to shift the E2 20 Å to accept the Ub/Ubl (Movie 1). Activated E2~Ub/Ubls can transfer the Ub/Ubl directly to a substrate lysine residue to form an isopeptide bond. My lab established that the SUMO E2 directly recognizes the substrate lysine and that the E2 harbors key residues required for maintaining an active site capable of ordering and deprotonating the lysine during conjugation, a mechanism conserved in other E2s. E3 ligases function to recruit a substrate and E2~Ub/Ubl into a complex to increase the rate of Ub/Ubl transfer and to provide substrate specificity. My lab determined the first structure for any quaternary complex between an E3, E2, Ub/Ubl, and substrate in studies that established a conformational activation model for E3-catalyzed E2~Ub/Ubl discharge, a feature shared with other RING-type E3 ligases.

Movie 1: The E1 activation cascade and transfer to E2.

A movie illustrating the E1 activation cascade based on crystal structures determined for the SUMO E1 before and during thioester bond formation and the Ub E1 before and during transfer to the E2. E1 adenylation domains are colored pink and blue, the FCCH domain is colored dark magenta, the ubiquitin-fold domain (UFD) is colored red, and the Cys domain is colored purple with the helix (red) containing the E1 catalytic cysteine (yellow sphere).

Early in the movie the E1 is in an open configuration. Ub (yellow) fades in with ATP and magnesium to form the Ub adenylate. The Cys domain then rotates to transit the catalytic cysteine into the adenylation-active site, a thioester bond is formed, and it returns to the open state to recruit another Ub (yellow), ATP and magnesium, and the E2 (green). Conformational changes in the E1 UFD domain bring the E1 and E2 catalytic cysteine residues together (yellow spheres) and the UFD returns to an open configuration, after which the E2~Ub departs. The cycle repeats after rotating the E1.

The movie was generated by Shaun Olsen, using several static structures as input to Chimera to generate individual structures (Pettersen, E.F., Goddard, T.D., Huang, C.C., Couch, G.S., Greenblatt, D.M., Meng, E.C., and Ferrin, T.E. 2004. Journal of Computational Chemistry 25:1605–1612). PyMol was used to generate individual frames (Delano, W. 2002. The PyMOL molecular graphics system. DeLano Scientific, San Carlos, CA. http://www.pymol.org).

Once a substrate is conjugated to a Ub/Ubl it can elicit responses that include recruitment of downstream factors to mediate signal transduction; however, little is known about how Ub/Ubl-modified targets are specifically recognized by their receptors. We investigated this by characterizing recognition of SUMO-conjugated proliferating cell nuclear antigen (PCNA) by Srs2, an antirecombinogenic helicase that prevents Rad51-dependent recombination during S phase. We determined that Srs2 harbors tandem receptor motifs that interact independently with PCNA and SUMO and that both motifs are required to specifically recognize SUMO-PCNA in vitro and in vivo. My lab is currently studying several related systems to establish models for receptor recognition during Ub/Ubl-mediated signal transduction.

RNA Processing, Quality Control, and Decay
RNA quality and abundance is regulated by surveillance and decay pathways. In eukaryotes, most RNA degradation proceeds 5'→3' or 3'→5'. The eukaryotic RNA exosome is a conserved multisubunit complex that is essential for maintaining the abundance and quality of RNA in the nucleus and cytoplasm by mediating 3'à5' RNA decay, quality control, and processing. The RNA exosome includes nine individually encoded protein subunits (Exo9) that comprise the exosome core (Rrp41, Rrp42, Rrp43, Rrp45, Rrp46, Rrp4, Rrp40, Csl4, and Mtr3). In the cytoplasm the exosome core associates with Rrp44, a processive 3'→5' exoribonuclease (Exo10), an enzyme that also harbors a second active site capable of endoribonuclease activity. In the nucleus the exosome core and Rrp44 interact with Rrp6, a distributive 3'→5' exoribonuclease (Exo11). In human cells there also appears to be a nucleolar exosome composed of the core and Rrp6 (Figure 2).

General messenger RNA (mRNA) decay can be initiated by removing the poly(A) tail, followed by decapping and 5'→3' decay, or by RNA exosome-mediated 3'→5' decay. The RNA exosome is also required for degradation of cryptic unstable transcripts (CUTs) that form through bidirectional transcription at promoters, degradation of transcripts targeted by small interfering RNAs (siRNAs) after endonucleolytic cleavage by RNA-induced silencing complex (RISC), and RNAi-induced heterochromatin spreading at centromeres. As a quality control machine, the nuclear exosome degrades misfolded or defective RNAs such as aberrant transfer, ribosomal, small nuclear, small nucleolar, and pre-mRNAs. In contrast, the exosome also functions in 3'-end processing during maturation of nuclear precursors of these RNAs. In the cytoplasm the exosome is a component of several mRNA surveillance pathways, including nonsense-mediated decay, nonstop decay, and no-go decay.

Movie 2: Structure of the human exosome core.

The movie illustrates the architecture of the nine-subunit human exosome core, with each of the six unique RNase PH-like proteins fading in, followed by capping of the six-subunit ring with the three unique proteins that have S1 and K homology (KH) domains (subunits colored as in Figure 2). The movie ends with a view looking down the central channel. The central channel and exosome core are required for RNA interactions and for modulating the activities of Rrp6 and Rrp44 (not shown).

Christopher Lima

We pioneered strategies to reconstitute 9-, 10-, and 11-subunit RNA exosomes from budding yeast and human to determine their architectures and activities. Our studies revealed the first structure for a eukaryotic nine-subunit exosome core (Movie 2) and that human and yeast exosome cores are devoid of catalytic activity, a surprising result given evolutionary relationships to bacterial PNPase and archaeal exosomes whose central channel harbors active sites for 3'→5' decay. Instead, the catalytically inert eukaryotic exosome core interacts with Rrp44 and Rrp6, presumably to regulate their RNase activity via subunit interactions or by filtering RNA through the central channel. We analyzed channel-occluding mutations and uncovered evidence that the central channel is essential and contributes to each of the known RNase activities of the budding yeast exosome. We also observed that the exosome core modulates Rrp6 RNase activity, that it inhibits Rrp44 activity, and that Rrp6 can activate Rrp44 RNase activity independent of Rrp6 catalytic activity. These and additional data suggest that Rrp6 and Rrp44 share at least one RNA-binding channel in the Exo11 complex and that Rrp6 and Rrp44 activities are regulated by association with the exosome core.

Our studies provide a framework for understanding exosome activities during RNA processing. Ongoing efforts in my lab are guided by the ideas that exosome RNase activities are modulated by the exosome core and that RNA substrates are targeted for destruction or processing by direct interaction with the exosome or through interactions with factors that deliver substrates to the exosome.

Grants from the National Institutes of Health provided partial support for some of these projects.

As of September 1, 2013

Scientist Profile

Investigator
Memorial Sloan-Kettering Cancer Center
Biochemistry, Structural Biology