Fig 1.
Exosome structure model and interactions.
(A) Overview of the structures of the TRAMP nuclear cofactor complex and the exosome. The main components of the exosome are schematically represented: the cap in red, the PH-ring in green, forming a contiguous channel (in lighter color) through which the RNA can be threaded. Active sites are indicated in Rrp6 (orange; exonuclease) and Rrp44 (dark blue; endonuclease (endo) and 3’ ->5’ exonuclease (exo)). The Rrp44 S1 RNA binding domain is represented in yellow and the channel to access the Rrp44 exonuclease site in light blue. Two conformations are illustrated: “channel-threading" of the substrate in which the exosome barrel channel is connected to the Rrp44 channel (left panel). A structural rearrangement can disconnect both channels to allow “direct-access” of substrates to the Rrp44 exonuclase site (right panel). Proteins analyzed by CRAC are in bold color. (B) Domain structure of the Rrp44-HTP fusion. From N-terminus to C-terminus, the following domains are indicated: PIN (PilT N terminus) domain harboring endonuclease activity, CSD (Cold-Shock Domain) RNA binding domain, RNB (RNase II ribonuclease) domain harboring exonuclease activity, S1 RNA binding domain and the HTP-tag (His6, TEV protease cleavage site, protein A). (C) Distribution of reads mapped to different RNA substrate classes recovered in CRAC datasets. Two biological replicates are shown for each protein.
Fig 2.
Targeting of the pre-rRNA 5’ external transcribed spacer 5’ ETS (RNAPI transcript) involves both channel threading and channel-independent pathways to access Rrp44.
(A) Northern analysis of RNAs coprecipitated with immunoaffinity purified (IP) active Rrp44-HTP (WT), Rrp44-exo-HTP (exo) or Rrp44-exo-S1-HTP (exo-S1), along with 2% input RNA. RNA species are detected with a probe hybridizing near the TSS of the 5’ ETS (+49–67, see panel B for location of the probe). Sybr safe staining for 5S rRNA is shown as loading control. (B) Distribution of reads across the 5’ ETS, recovered with Mtr4, Rrp6, Csl4, Rrp41 in an Rrp44-exo background, and Rrp44-exo and Rrp44-exo-S1, normalized to millions of mapped reads. Scale is linear. A diagram of the 5’ ETS and the 18S rRNA is also shown. (C) Model for 5’ ETS degradation. Following cotranscriptional cleavage of the pre-rRNA, the 5’ ETS is oligo-adenylated by TRAMP and targeted to Rrp44 through the channel. The 5’ ETS is subsequently released from the channel (possibly aided by Mtr4 activity) and subjected to new oligo-adenylation by TRAMP, before being targeted to Rrp44 through direct access.
Fig 3.
RNAPIII transcripts show differences in threading through the channel.
(A) RPMs (reads per million mapped reads) for each RNA species were averaged between two replicates of either Rrp44-exo (column 2) or Rrp44-exo-S1 (column 3) constructs and arranged by k-medians clustering (k = 4, column 1). Distributions of pre-tRNAs (blue), tRNAs (purple), 5S rRNA (yellow) and other non-coding RNAPIII transcripts (green) are indicated in column 4. Intron-containing pre-tRNAs are indicated in green in column 5. Transcripts discussed in the text are indicated in column 6. See also S3 Table. (B) Relative protein association of all RNAPIII transcripts from each cluster was calculated for Mtr4, Rrp6, Csl4, Rrp41, Rrp44-exo and Rrp44-exo-S1 in total RPM. Averages between two independent experiments are shown with standard deviation, except for Rrp41 where fewer reads were recovered and only the largest dataset is shown. (C) 2D scatter-plot comparing RPM across pre-tRNAs and tRNAs recovered with Rrp44-exo and Rrp44-exo-S1. (D-E) Northern analysis of RNAs coprecipitated with Rrp44-HTP (WT), Rrp44-exo-HTP (exo) or Rrp44-exo-S1-HTP (exo-S1), along with 2% (D) or 1% (E) input RNA, probed for RNAPIII transcripts: U6 snRNA, scR1, 5S rRNA, RPR1 (D) or tRNAPro(UGG) (E). Sybr safe staining for 5S rRNA is shown as loading control. Asterisks indicate previously reported truncation products that are known exosome substrates.
Fig 4.
Rrp44 is involved in two distinct tRNA processing pathways.
(A-F): Metagene analysis of binding across tRNAs for Rrp44-exo (A), Rrp44-exo-S1 (B), Csl4 (C), Rrp41 (D), Mtr4 (E) and Rrp6 (F), all in an Rrp44-exo background. Upper plots show read distributions across all tRNA genes ordered by length and aligned by the 3’ termini of mature tRNAs. Numbers above color scale box indicates the maximum number of hits recovered in individual genes. Green lines indicate the 5’ and 3’ boundaries of the mature tRNAs. Longer genes at the foot of the columns are intron-containing. Total reads are presented in lower graphs in each panel. (G) Metagene analysis of binding across tRNA genes for Rpo31 (RNAPIII subunit) (black), Rrp44-exo (blue) and Rrp44-exo-S1 (yellow) aligned to mature tRNA 5’ ends. Dashed lines in the Rrp44 samples indicate binding profiles specifically for reads that include 3’ oligo(A) tails absent from the genomic sequence. (H) Model for the degradation of 5’ regions of tRNAs: Aborted transcripts released by RNAPIII pausing are oligo-adenylated by TRAMP and targeted to Rrp44 through the exosome channel. (I) Model of 3’ processing of tRNAs: Following transcription termination, 3’ extended pre-tRNAs directly access Rrp44.
Fig 5.
RNAPII transcripts show differences in threading through the channel and direct access to Rrp44.
(A) Clustering based on reads per kilobase per million total mapped reads (RPKM) for each transcript for top 1000 mRNAs, top 200 SUTs, top 200 CUTs, 75 snoRNAs and 4 snRNAs. Hits were averaged between two replicates of either Rrp44-exo (column 2) or Rrp44-exo-S1 (column 3) constructs and arranged by k-medians clustering (k = 4, column 1). Location of mRNAs (grey), snRNAs (green), snoRNA (dark red), CUTs (blue) and SUTs (orange) were indicated in column 4. See also S4 Table. (B) Association of all RNAs from each cluster with Mtr4, Rrp6, Csl4, Rrp41, Rrp44-exo and Rrp44-exo-S1 in total RPKM. Averages between two independent experiments are shown with standard deviation, except for Rrp41 where fewer reads were recovered and only the largest dataset is shown.
Fig 6.
snoRNAs use both channel threading and direct access to Rrp44 for processing.
(A, B) Metagene analyses of all box H/ACA snoRNAs (A) or all box C/D snoRNAs (B) aligned by the 3’ end of the mature snoRNA region. Two independent experiments for Rrp44-exo (blue) and Rrp44-exo-S1 (yellow) are shown. (C, D): Distribution of reads across the box C/D snoRNAs U14 (C) and U3 (D), recovered with Mtr4, Rrp6, Csl4, Rrp41 in the Rrp44-exo background, and Rrp44-exo and Rrp44-exo-S1, normalized by millions of mapped reads. Scale is linear. (E-F) Northern analysis of RNAs coprecipitated with wild type Rrp44-HTP (WT), Rrp44-exo-HTP (exo) or Rrp44-exo-S1-HTP (exo-S1), along with 2% (E) or 1% (F) input RNA, probed for the box C/D snoRNAs U14 (E) or U3 (F). Sybr safe staining for 5S rRNA is shown as a loading control.
Fig 7.
Cytoplasmic processing of mRNAs is affected by the Rrp41-channel mutation.
(A) Distributions of RNA classes in mapped reads recovered with Rrp44 in strains expressing wildtype Rrp41 (left columns) or the Rrp41-channel mutant that is predicted to partially occlude the central channel of the exosome (-channel, right columns). Two biological repeats are shown for each strain. (B-C) Metagene analysis of binding across mRNA genes for Rrp44 in strains expressing wild type Rrp41 (blue) or the Rrp41-channel mutant (green) aligned to the transcription start site (TSS), for all reads (B) or only reads that include non-encoded 3’ oligo(A) tails (C), normalized per millions mapped reads. Data from two biological repeats were averaged for each analysis. (D-F) RPKMs for each RNA species were averaged between two replicates of either Rrp44 with wild type Rrp41 or the Rrp41-channel mutant construct and displayed on a 2D scatter plot for top 200 mRNAs (D), top 200 CUTs (E) or top 200 SUTs (F). Species above the diagonal line are predicted to be strongly subject to nuclear degradation. See also S5 and S6 Tables. (G) Mtr4 binding (RPKM) across mRNAs in function of ratio of Rrp44 binding between strains expressing wild type Rrp41 and the Rrp41-channel mutant. Mtr4 preferentially binds mRNAs not affected by channel mutation, consistent with nuclear degradation. (H-I) Distribution of reads recovered with Mtr4 or Rrp44 (with wild type Rrp41 or the Rrp41-channel mutant) across the TDH3 gene (H), targeted less in Rrp41-channel strains, and RPS14B, which is not sensitive to channel mutation, normalized to millions of mapped reads. Scale is linear.