Rrp6: Integrated roles in nuclear RNA metabolism and transcription termination

Abstract

The yeast RNA exosome is a eukaryotic ribonuclease complex essential for RNA processing, surveillance, and turnover. It is comprised of a barrel-shaped core and cap as well as a 3'-5' ribonuclease known as Dis3 that contains both endo- and exonuclease domains. A second exonuclease, Rrp6, is added in the nucleus. Dis3 and Rrp6 have both shared and distinct roles in RNA metabolism, and this review will focus primarily on Rrp6 and the roles of the RNA exosome in the nucleus. The functions of the nuclear exosome are modulated by cofactors and interacting partners specific to each type of substrate. Generally, the cofactor TRAMP (Trf4/5-Air2/1-Mtr4 polyadenylation) complex helps unwind unstable RNAs, RNAs requiring processing such as rRNAs, tRNAs, or snRNAs or improperly processed RNAs and direct it toward the exosome. In yeast, Rrp6 interacts with Nrd1, the cap-binding complex, and RNA polymerase II to aid in nascent RNA processing, termination, and polyA tail length regulation. Recent studies have shown that proper termination and processing of short, noncoding RNAs by Rrp6 is particularly important for transcription regulation across the genome and has important implications for regulation of diverse processes at the cellular level. Loss of proper Rrp6 and exosome activity may contribute to various pathologies such as autoimmune disease, neurological disorders, and cancer. WIREs RNA 2016, 7:91-104. doi: 10.1002/wrna.1317 For further resources related to this article, please visit the WIREs website.

Figure 1. Schematic representation of the S. cerevisiae nuclear exosome and its known cofactors

The core barrel structure (also known as Exo9), in green, consists of nine subunits: Rrp41, Rrp42, Rrp43, Rrp45, Rrp46, and Mtr4 (the six RNase PH-like proteins), and Rrp4, Rrp40, and Csl4 (the three S1/KH cap proteins). The nuclease Dis3 associates with the core barrel in both the cytoplasm and the nucleus. This complex is also called Exo10. Rrp6 only joins the core exosome in the nucleus, in a complex known as Exo11. The cofactor Rrp47 (dark red) binds to Rrp6 and structured RNA and improves the stability of Rrp6. The TRAMP complex (orange) binds to the surface formed by Rrp6/Rrp47 to thread RNA substrates through the exosome. Nrd1 and its binding partner Nab3 (gray) interact with Rrp6, both together and independently of one another to coordinate termination and processing of short (<1000 nt) RNAs. The cap binding complex (purple) interacts with Rrp6 during co-transcriptional processes in humans. In yeast, the cap binding complex copurifies with Nrd1 and Nab3, which may lead to an indirect interaction with Rrp6. Mpp6 (dark green) is a general nuclear exosome cofactor involved in a number of exosome-dependent mechanisms such as processing and degradation of a RNA arising from rDNA arrays.

Figure 2. Model of Nrd1-dependent transcription termination

In yeast, Rrp6 may have a direct role in NNS-dependent termination of short RNAs through protein-protein and protein-RNA interactions. Nrd1 binds S5-P on the RNAPII CTD, and Nrd1 and Nab3 bind the nascent RNA. The mechanism by which Nrd1 causes termination is not known, but may involve an interaction with Rrp6. Sen1 unwinds RNA:DNA hybrids in a 3’–5’ direction relative to the nascent RNA facilitating RNAPII termination. The TRAMP subunit Trf4 binds Nrd1, releasing it from the CTD (indicated as a red ‘X’). TRAMP adds a short polyA tail to the RNA, targeting it for degradation by the exosome. Rrp6 interacts with Nrd1, likely still bound to the RNA to inhibit complete degradation of stable transcripts such as snRNA. RNAPII must then be removed from the DNA and proteins bound to both the RNA and RNA must be released through unresolved mechanisms.

Figure 3. Rrp6 is required for proper Nrd1-dependent termination at some short RNAs

RNA sequencing and Rpb3 ChIP-exo data at snR762 in wild type and rrp6Δ. Data previously published in . (A) Graphical representation of strand-specific RNA-seq reads mapped to snR62 region. Only reads mapped to the snR62 strand are shown because reads mapped to the other strand are minimal. The location and direction of transcription for all analyzed annotations on the strand of interest are diagrammed below the graphs to scale. Processed length of snRNAs and mRNAs are in black, snRNA-extended transcripts, including pre-snRNAs and termination read-through products, are in green (labeled “ETs”), NUTs are in aqua, and arrows indicate direction of transcription. (B) Rpb3-FLAG localization as determined by ChIP-exo sequencing reads mapped to the same region and aligned to (A). Wild-type normalized read counts are in black, and rrp6Δ are in orange. The region of increased RNAPII localization in rrp6Δ is indicated by the black arrows.

Figure 4. Rrp6 could play an indirect role in the regulation of Nrd1-dependent termination

In the speculative RNA-dependent model, a theoretical cryptic lncRNA that is usually degraded by Rrp6 may be stabilized in its absence. This trans-acting RNA may then sequester one or more proteins required for Nrd1-dependent termination. This subnuclear trapping may be similar to the mechanism by which NEAT1 sequesters RNA-binding proteins in paraspeckles, effectively inhibiting their activity without decreasing their overall abundance. Nrd1 and Nab3 would be reasonable candidates for such a mechanism considering their RRM domains, or another currently unidentified protein could also be affected.

Figure 5. The nuclear exosome may facilitate rescue of backtracked RNAPII

Dis3 and the core exosome have been found to facilitate termination of backtracked RNA, but Rrp6 did not play a significant role in terminating backtracked mRNA transcripts in S. pombe. Rrp6 has been found to play a more substantial role in termination of cryptic antisense transcripts originating within gene bodies and certain snRNAs , so it is possible that Rrp6 assists backtracking polymerase in a similar model at these transcripts. In this speculative model, the 3’ end of the RNA is in the active site when RNAPII is actively transcribing, protected by RNAPII, and inaccessible to Rrp6. When polymerase backtracks in response to an obstacle in the DNA or improperly incorporated nucleotide, the 3’ end of the RNA emerges through a ‘backtrack site’ in RNAPII and can be accessed by Rrp6 .

Figure 6. Multiple mechanisms by which Rrp6 processes or degrades its many RNA substrates

(A) The 18S, 5.8S, and 25S rRNAs are transcribed as one molecule and cleaved and processed by several nucleases. Specifically, Rrp6 is required for processing of the 3’ end of the pre-5.8S product. (B) Improperly processed tRNAs in the nuclease are targeted by the TRAMP complex which adds a short polyA tail and directs the substrate to the exosome for complete degradation by Rrp6. (C, left) Exo11 interact directly with the spliceosome to degrade introns co-transcriptionally. (C, right) Rrp6 also degrades mRNAs that cannot be exported and accumulate at the transcription site when the accumulation is due to improperly processed 3’ ends or inhibition of nuclear export machinery. (D, left) Rrp6 is required for proper termination by Nrd1. The mechanism by which Nrd1 causes termination is not known. It may sometimes require an interaction with Rrp6 to properly terminate, including release of DNA and RNA from RNAPII and the termination factors. (D, right) Rrp6 processing the 3’ end of snRNAs terminated in a heterogeneous “zone.” The extended 3’ ends of the pre-snRNA are trimmed back by Rrp6. (E) Cryptic Unstable Transcripts (CUTs) and unstable nuclear lncRNAs are both targeted to the Exo11 by the TRAMP complex.