Human 9-Subunit Exosome

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Human 9-Subunit Exosome

Michael Itschner II '18 Nick Vitale '19

I. Introduction

The degradation of cellular RNA can happen via two different pathways, but both are initiated by the shortening of the polyadenylated tail. In one pathway, degradation of 5' to 3' RNA is done by a 5' to 3' exoribonuclease, such as Xrn1. The second pathway of degradation occurs in a 3' to 5' direction by a 3' to 5' multisubunit exoribonuclease, such as an exosome, or the human 9-Subunit Exosome (hExo9) in this case. Exosomes can also process small nuclear RNA's (snRNA) and ribosomal RNA through their phosphorolytic chamber, in which only degraded single-stranded substrates are able to fit. hExo9 demonstrates little to no RNase activity for most RNA substrates, except for regions of the RNA that have rich AU substrates. Exosomes are also found to be regulated by its association with other complexes such as the TRAMP complex, which primes RNA for degradation.
There are several forms of exosome, 9, 10, and 11 subunit complexes, all of which have different activity levels. Of these three, the 9-subunit Exosome is considerably less active than the 10 subunit versions. Currently, hExo9 consists of nine polypeptide subunits that are composed of 2213 aa out of a possible 2575 aa. The hExo9 consists of two large domains, the PH ring and the S1 proteins, where the contacts between them are highly conserved to ensure optimal interactions with the correct subunit interactions. This conservation suggests that the formation of a stable complex, involves all nine subunits.

II. General Structure

The exosome is made of two domains: the Plekstrin Homology (PH) domain and the S1 domain. The consists of three heterodimers and and is the more active RNase domain that forms a large ring-like complex. The three heterodimers of the PH domain ring consists of six polypeptide proteins of hRrp41/hRrp45, hRrp46/hRrp43, and hMtr3/hRrp42. Each PH domain polypeptide makes contacts to its dimer partner through conserved amino acid sequences. These conserved regions are critical to ensure correct dimerization of the subunits to form the hExo9 complex easily.

The most active dimer is the hRrp41/hRrp45 and is held together by contacts between conserved sequences: hRrp41's 195-215 aa and hRrp45's 230-250 aa. The same specific regions in hRrp46/hRrp43 and Mtr3/hRrp42 interfaces show similar conservation and account for the dimer's stability. The surfaces between the dimers' favor interactions with the correct partner and do not form strong interactions with the other polypeptides, resulting in correct dimer pairings. Each dimer pairing has similar contacts across the board, but one particular PH polypeptide has some interesting interactions. hRrp45's wraps around hRrp46 and hRrp43 through an extended helix of 180 amino acids that is highly conserved in eukaryotes. It begins by forming an interface with hRrp46 before wrapping around the two other subunits. It is also possible that this tail also makes contacts with Mtr3 but the last 120 C-terminal aa have not yet been determined. There is confidence that the tail is up to 302 aa long since Met298 was determined to be apart of the tail through selenium substitution. This contact is especially interesting because no other PH dimer contacts another PH dimer.

The consists of three separate proteins that sit on top of and form bridges between the heterodimers of the PH domain. These separate poly-peptides hRrp40, hRrp4, and hCsI4 are necessary for assembly and stability of the entire hExo9 complex. The S1 proteins sit on top of the PH heterodimers and around the open hole in the center of the PH domain. They have several conserved surfaces that are exposed to outside solvents, while the interface between the two domains does not have any notable conservation. These conserved sequences are interesting because they flank the central channel to the active site of the complex. In turn, the proposed route that the substrate undertakes to enter the protein has to pass through the S1 domain before it is processed by hExo9.

III. PH-S1 Domain Interactions

Extensive contacts are formed between the subunits of the S1 and the heterodimers of the PH domains. The interaction of the domains occur on the top of the PH ring and the bottom of the S1 domain. Just like the interactions within the dimers of the PH ring, the conserved surfaces have evolved to favor their correct pairings. hRrp40 makes contacts with hRrp45 and hRrp46, hRrp4 contacts hRrp41 and hRrp42,, and hCsI4 contacts hMtr3 and hRrp43 . The S1 subunits make contacts with the dimers of the PH-ring with their N-terminal domains: hCs14, hRrp4, and hRrp40 make contacts with hMtr3, hRrp41, and hRrp46 PH subunits respectively. These interactions between the two domains is key to the hExo9 stability and function. An attempt to isolate a stable 6 subunit PH ring without the presence of the S1 domain proteins failed. This provided strong support that the S1 subunits hold together the PH heterodimers and are responsible for the complexes stability

IV. Active Site

Of the three heterodimers, the hRrp41/hRrp45 dimer of the PH domain is the only one that exhibits processive phosphate-dependent activity that produces RNA molecules of 4-5 nucleotides long. This dimer houses the phosphorolytic active site and aids in the binding of RNA. Mutations in either of the dimer subunits lead to no detectable activity for RNA binding or degradation. It was determined that the hExo9 only has one phosphorolytic active site, which is the hRrp41/hRrp45 dimer and does not contain any other sites of similar activity.

The active site of hExo9 is not very active in comparison to other exosome. However the hExo9 readily degrades RNA that has rich AU nucleotide repeats. The hExo9 is also selective for what kind of RNA or any other kind of substrate it degrades. The that is formed by the S1 domain proteins, prevent any substrate that is too large or has too much structure from entering it. The types of RNA that are able to enter the complex are those that do not have any secondary structure and have been partially degraded prior to entry. It was presumed that the minimum length of RNA that is needed to observe activity in the active site would be determined by the height of the exosome.

The RNA would enter the complex between the hRrp41/hRrp45 dimer and then continue to go through the center channel of the complex between the S1 proteins . hRrp41's Arg94 and Lys95 in conjunction with hRrp45's Arg104, Arg108, and Arg111 all have been found to be necessary in binding the exosome's substrate, single stranded RNA . hRrp41's Asp130, Thr133, and Tyr134 have been found to be a conserved phosphate binding site . Phosphate binding is key to the action of the exosome as the bound phosphate would aid to cleave the phophodiester bonds in the RNA's backbone.

V. References

Allmang, C., Kufel, J., Chanfreau, G., Mitchell, P., Patfalski, E., and Tollervey, D. (1990). Functions of the exosome in rRNA, snoRNA and snRNA synthesis. EMBO J. 18, 5399-5410.

Bank, R. P. D., Greimann, J.C., & Liu, Q. (2006). RCSB PDB - 2NN6: Structure of the human RNA exosome composed of Rrp41, Rrp45, Rrp46, Rrp43, Mtr3, Rrp42, Csl4, Rrp4, and Rrp40 structure summary page. Cell(Cambridge,Mas,), 127, 1223-1237.

Liu, Q., Greimann, J. C., & Lima, C. D. (2006). Reconstitution, activities, and structure of the eukaryotic RNA exosome. Cell, 127(6), 1223-1237.

PDB-101: Exosomes. (2007, October). Retrieved December 9, 2016, from https://pdb101.rcsb.org/motm/86
Shen, V., & Kiledjian, M. A view to a kill: Structure of the RNA exosome. Cell, 127 (6), 1093-1095.

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