The RNA exosome composed of Rrp41, Rrp45, Rrp46, Rrp43, Mtr3, Rrp42, Csl4, Rrp4, and Rrp40 (PDB ID: 2NN6) from Homo sapiens is a multisubunit 3’ to 5’ exoribonuclease complex. Human RNA exosome is a known catalyst involved in the degradation of cellular mRNA (1).  In mRNA degradation, the exosomal activity has been proposed to be regulated by the Ski2p/Ski3p/Ski8p complex and Trf4p/Air2p/Mtr4p polyadenylation complex  (TRAMP) (2,3). The RNA exosome has also been found to be involved in the processing of both of small nucleolar (snoRNA), small nuclear (snRNA), and ribosomal RNAs (rRNA) (4). 

Mutations in the SKI genes were originally identified because of overexpression of the gene product in yeast double-stranded RNA virus (2). Initially, the homology of the Ski6p/Rrp41p protein to the Escherichia Coli 3’ to 5’ exoribonuclease RNase PH suggested the involvement of exosomes in the degradation of mRNA (2). The SKI genes involvement also implies that exosome activity is regulated by the Ski2p/Ski3p/Ski8p complex (2).  While on the other hand, TRAMP is presumed to prime structured RNA through polyadenylation to assist interaction with the exosome (3). The exosome complex of 3’-5’ exonucleases is known to aid with quality control and has been observed to quickly degrade RNA-protein complexes in vivo (3). However, purified exosome displays weak activity in vitro, indicating that there are required activating cellular cofactors for full function (3). 

There is no definite reason why there are nine exonucleases present in the human RNA exosome complex. It is possible that multiple enzymes may function to facilitate the processing different RNA substrates (snoRNA, snRNA, and rRNA), allowing one exosome to serve multiple functions in the interest of genetic economy. Also, different enzymes in the complex may be more active on different substrates (4).

Human RNA exosome in the asymmetric unit (286 kDa) containing all exosome subunits (Rrp41, Rrp45, Rrp46, Rrp43, Mtr3, Rrp42, Csl4, Rrp4, and Rrp40) was crystallized at 18°C by vapor diffusion. Additives, such as glycerol, were found to sometimes increase crystal quality. The crystals were allowed to grow for 20 days and the crystalline structure was determined by x-ray diffraction (1). 

The molecular weight and isoelectric point of the human RNA exosome complex were calculated with Expasy to be 286235.15 Da and 6.06  (5). The near-neutral nature of the complex reflected in the number of acidic and basic regions found in the protein complex. Rrp41, Rrp45, Rrp46, Rrp43, Mtr3, Rrp42, Csl4, Rrp4, and Rrp40 individually had theoretical weights of 26.83 kDa, 39.41 kDa, 25.48 kDa, 30.29 kDa, 28.27 kDa, 33.48 kDa, 23.1 kDa, 34.59 kDa, and 31.23 kDa, respectively (1). Each of the individual subunits is comprised of polypeptide strands containing beta-strands, beta bridges, and alpha helices (1).  Human nine-subunit cytoplasmic exosome (hExo9) is comprised of a ring of cap proteins bound to the top of the RNase PH-like proteins (10). hExo9 complexes were reconstituted containing mutations in either hRrp41 at residue positions T133A and Y134L or hRrp45 at residue positions R104N and R111N. Both of these mutational events were found to abolish 3’ to 5’ exoribonuclease activity, suggesting that hExo9 has one phosphorolytic active site (hRrp41/Rrp45) (1). 

Position-Specific Iterated Basic Local Assignment Search Tool (PSI-BLAST) and the Dali Server were used to reveal proteins that have a similar primary and tertiary structure with human RNA exosome. The PSI-BLAST program is an online tool that searches for proteins that are similar to the primary structure of the protein query. PSI-BLAST returns a list of proteins with E-values, which indicate how similar the sequence homology is to the query protein, the human RNA exosome (6). The E-value is calculated by overlapping the sequence of human RNA exosome to all other proteins in the database and to find gaps. The fewer gaps a particular protein has to the query, the lower the E value. An E value below 0.05 indicates that the two proteins are significantly similar in primary structure. While the Dali server compares the three-dimensional structures of proteins to reveal functional clues that indicate folding similarities. In particular, Dali utilizes the sum-of-pairs method to compare intramolecular distances (7). Proteins of interest are assigned Z-scores depending upon tertiary structure similarity. Z-scores greater than 2 signify that there is similar tertiary folding between the two proteins. 

The bioinformatic analysis of RNA exosome from Homo sapiens revealed that chain A of the crystal structure of an 11-subunit eukaryotic exosome complex bound to RNA (PDB ID: 4IFD) from Saccharomyces cerevisiae has both similar primary and tertiary structures. The two proteins share an E-value of 7.0e-64 and Z-score of 33.3 (8,9). The 11-subunit RNA exosome complex of Saccharomyces cerevisiae (440 kDa) is composed of 10 subunits (Exo-10) bound to a carboxyl terminal region of Rrp6 and duplexed to RNA with a 3’-overhang of 31 ribonucleotides (10). Rrp6 folds into two different polypeptide stretches that bind to S1/KH and RNase proteins.The first polypeptide stretch(residues 532-557) forms an alpha helix that has a binding affinity to the subunits CsI4 and Mtr3. The second polypeptide stretch ( residues 565-599) has a small beta-hairpin and alpha-helix that contact Mtr3, Rrp43, and CsI4 (10). 

In hExo9, all of the subunits contain amino acid changes in their proposed active site cause them to become inactivated (1). All Exo-9 subunits are known to contribute to the binding of phosphate groups along the RNA backbone (10). However, exosomal activity is only initiated after an additional subunit, Rrp44, is added to the complex (10). This exo-10 complex can then recruit additional factors and regulators that can act in the cytoplasm or nucleus (3). In the 11-subunit eukaryotic exosome complex, Rrp44 has been found to adopt a closed conformation that captures the 3’-end of the RNA that exits Exo-9. Rrp44 is a unique subunit in the sense that it contains both an endoribonuclease and exoribonuclease region (10). Rrp44 specifically binds to the RNase PH barrel on the opposite side of the biomolecule as the S1/KH ring (10). 

RNA to be degraded enters the RNA exosomal chamber in an unwound conformation, similar to that when entering a proteasome (10). When the RNA enters the Exo9 complex, it has been observed to traverse about half of the barrel-like structure before being pushed to the side (10). The RNA in the Exo9 complex follows a similar binding path as that found in prokaryotic exosome-like complexes that lead to a phosphorolytic site (10). In yeast it has been observed that in the absence of such a phosphorolytic site, the RNA continues uninterrupted within the Rrp44 nuclease, then curls up on the side of Exo-9, and grabs the 3’-end of the RNA (10). The mechanisms of exosome complexes are surprisingly conserved from prokaryotes to eukaryotes (10). 

The activities profiled in both the reconstituted human and yeast exosomes suggest that many of the subunits play non-catalytic roles in the processing and degradation of RNA. It was previously believed and modeled that some of the exosomal subunits did not play a vital role in these molecular processes. Although yeast exosome is known to function without its Rrp6 subunit, all other exosome subunits have been determined to be essential for the larger biomolecular processing and degradation of RNA (1). Non-catalytic subunits may play important roles in the recruitment of RNA specific substrates; however, further research is required before this conclusion can be made. Continued research on exosomal function in different organisms will help to elucidate the full specific molecular function for each of the exosomal subunits, the conservative nature of the RNA exosome, and the cofactors associated with in vivo activity.