1. Volume 64, Issue 3, Pages 520-533 (November 2016)
Identification of a Nuclear Exosome Decay Pathway for Processed Transcripts 
Nicola Meola, Michal Domanski, Evdoxia Karadoulama, Yun Chen, Coline Gentil, Dennis Pultz, Kristoffer Vitting-Seerup, Søren Lykke- Andersen, Jens S. Andersen, Albin Sandelin, Torben Heick Jensen 
Molecular Cell 
Volume 64, Issue 3, Pages (November 2016)
DOI: /j.molcel
Copyright © 2016 Elsevier Inc. Terms and Conditions

2. Molecular Cell 2016 64, 520-533DOI: (10.1016/j.molcel.2016.09.025)
Copyright © 2016 Elsevier Inc. Terms and Conditions

3. Figure 1 ZFC3H1 Forms a Stable, NEXT-Independent Complex with hMTR4
(A) Scatterplot displaying specific proteins identified in triplicate hMTR4-LAP ACMS experiments from HeLa Kyoto cells conducted in the presence or absence of RNase A/T1. Results are plotted as RNase A/T1 resistance (ratio of the relative protein abundance [total peptide intensity divided by molecular weight and normalized to hMTR4-LAP data] between RNase+ and RNase− experiments) out the x axis, and relative protein abundance from RNase− samples up the y axis. Error bars denote SDs calculated from triplicate experiments. Note disruption of both axes to accommodate all data. Proteins relevant to this study are name labeled and indicated with black dots. Exosome-related factors are shown as red squares. Gray dots represent proteins specifically co-purifed with hMTR4-LAP and are grouped into five categories highlighted in Figure S1. The entire dataset is listed in Table S1.
(B) Immunofluorescence microscopy analysis of HEK293 Flp-In T-Rex cells stably expressing ZFC3H1-3xFLAG. Cells were stained with α-FLAG antibody and DAPI, to visualize nuclei, as indicated. Dashed lines represent cell outlines (drawn based on the DIC image). Shown examples are representative for the entire cell population.
(C) SDS-PAGE analysis followed by Coomassie staining of the eluates from RBM7-LAP or ZFC3H1-LAP purifications as indicated. Both proteins were affinity isolated from HeLa Kyoto cells using α-GFP antibodies and eluted with TEV protease. Migration of molecular-weight markers is indicated to the left of the image. Band identities were deduced by migration relative to size markers and by parallel western blotting analysis.
(D) Western blotting analysis of indicated proteins co-purifying with hMTR4-LAP, RBM7-LAP, and ZFC3H1-3xFLAG fusions. Input and eluate samples were probed with α-hMTR4, α-ZFC3H1, α-ZCCHC8, and α-RBM7 antibodies as indicated. Note the co-detection of hMTR4-LAP and RBM7-LAP with their endogenous (endo) counterparts. Due to the minor size difference, ZFC3H1-3xFLAG and endogenous ZFC3H1 proteins could not be distinguished.
See also Figure S1.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 2 ZFC3H1 Connects hMTR4 to PABPN1
(A) Scatterplot displaying specific proteins identified in triplicate ZFC3H1-3xFLAG ACMS experiments from HEK293 cells conducted and denoted as in Figure 1A. Gray dots represent proteins that are grouped into five different categories highlighted in Figure S2. The entire dataset is listed in Table S2. For presentation purposes, an abundance cutoff was made at 200, which excluded eight proteins from the scatterplot (see Table S2).
(B) Validation of selected MS results from (A) by AC and western blotting analysis. α-FLAG beads were used to capture ZFC3H1-3xFLAG and GFP-3xFLAG protein complexes, which were either treated (+) or not (−) with the RNase A/T1 mix. Released (R) material from these reactions and their 1xLDS eluted (“E”) counterparts were subjected to SDS-PAGE and western blotting analysis using α-ZC3H18, α-hMTR4, α-ARS2, α-CBP80, α-PABPN1, and α-FLAG antibodies as indicated. The migration of molecular-weight markers is indicated to the left of the image.
(C) Shown on left, western blotting analysis of hMTR4-LAP purifications performed using extracts from HeLa Kyoto cells depleted of ZCCHC8, ZFC3H1, or both proteins as indicated. Control cells (CTRL) were treated with siRNAs against Luciferase RNA. Captured material was eluted using 1xLDS loading buffer. Obtained input and eluate samples were probed with α-ZFC3H1, α-ZCCHC8, α-hMTR4, α-PABPN1, and α-RBM7 antibodies as indicated. Note that for input and eluate samples, α-hMTR4 signals derive from endogenous and hMTR4-LAP proteins, respectively. Asterisk depicts detection of cross-contaminating protein. Shown on right, cartoon representations of interpretation of the results from left. Crossed out names correspond to depleted proteins.
See also Figure S2.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 3 PAXT and NEXT Are Functionally Separated
(A) Western blotting analysis using extracts from HeLa Kyoto cells depleted for RRP40, RBM7, ZCCHC8, ZFC3H1, or PABPN1 as indicated. Control cells were treated with siRNAs against GFP.
(B) RT-qPCR analyses of expected PAXT substrates (spliced RNAs from snoRNA-host genes, left) and NEXT substrates (PROMPTs, right) using total RNA harvested from color-coded, factor-depleted samples. Design of qPCR amplicons is illustrated schematically above each panel. RT reactions were primed by random hexamer primers.
(C) UV-RNA immunoprecipitation (UV-RIP) analysis. Shown on left, western blotting analysis using α-GFP antibody to detect the presence of LAP, RBM7-LAP, and ZFC3H1-LAP proteins in the conducted UV-RIP experiments. Input samples represent 0.5% of protein extracts before IP. Shown on right, RT-qPCR analysis as in (B) of indicated RNAs co-eluted with LAP, RBM7-LAP, and ZFC3H1-LAP proteins. RT-qPCR levels from eluate samples were normalized to their respective input samples and to the control LAP sample (see Experimental Procedures).
(D) UV-RIP analysis in PABPN1 depletion conditions. Both western blotting analysis (left) and RT-qPCR assays (right) were performed as in (C). LAP-tagged and ZFC3H1-LAP-tagged cells were treated with luciferase (−) or PABPN1-specific (+) siRNAs as indicated. For westerns, input samples represent 1% of protein extracts before IP.
For statistical analysis, all data are displayed as mean values with error bars denoting SDs (n = 3 biological replicates). In (C) and (D), asterisks indicate detection of proteins of unknown origin.
See also Figure S3.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 4 Global Analysis of NEXT versus PAXT Target Preferences
(A) PCA based on sensitivity to the indicated depleted component as measured by RNA-seq across 19,252 GENCODE transcripts (see Experimental Procedures). Axes show principal components (PC) 1 and 2; percent of variance explained by each PC is indicated. Left and right panels show BrU and total RNA samples, respectively. Gray circles mark NEXT and PAXT components.
(B) Average sensitivity to indicated color-coded component depletions of RNA, expressed in regions 3 kb upstream and downstream of PROMPT-transcription initiation (top row) and eRNA-transcription initiation (bottom row). The y axes show the average sensitivity and the x axes show the nucleotide position relative to the respective PROMPT TSSs or enhancer midpoints. BrU and total RNA data are shown on the left and right, respectively. Forward-strand and reverse-strand signals are as indicated by black arrows. Data are displayed with 95% confidence intervals. Heatmap insets show the overall log2 sensitivity of individual PROMPTs or eRNAs (rows) for each component depletion (columns), sorted by descending RRP40 sensitivity. In this analysis, PROMPT and eRNA signals were collected from 1 kb TSS-downstream regions on the relevant strand. Red arrows indicate potential PAXT substrates.
See also Figure S4.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 5
Biochemical Properties of RNAs Determine NEXT versus PAXT Sensitivity
(A) PAXT and NEXT sensitivities of RNAs from BrU libraries are plotted as functions of their expression levels. The y axis shows the log2 PAXT:NEXT ratio (see text for details) and the x axis shows the log2 average RNA-seq support (TPM) from all analyzed libraries. Each dot represents one RNA target region as defined by color coding. Ellipses indicate the general location of transcript classes, colored accordingly. RNA targets with log2 PAXT:NEXT ratios above 0.5 or below −0.5 were assigned as PAXT and NEXT substrates, respectively, as indicated by the dashed lines.
(B) Length distributions of BrU RNAs preferentially targeted by PAXT or NEXT. The x axis shows median RNA lengths for each analyzed target, as inferred by TIFseq, in log10 scale. The y axis shows length distributions of RNAs defined as PAXT or NEXT targets in Figure 5A. p value is calculated by two-sided Mann-Whitney test.
(C) Polyadenylation status of RNAs preferentially targeted by PAXT or NEXT in BrU RNA libraries. The x axis shows p(A)+:(pA− + pA+) TPM ratios. The y axis shows density of the distribution of this ratio for PAXT and NEXT targets from Figure 5A. p value is calculated by two-sided Mann-Whitney test.
(D) RT-qPCR analyses of total RNA harvested from HeLa Kyoto cells treated with cordycepin for 3 or 6 hr as indicated. Untreated cells are depicted as “Cordy 0.” qPCR amplicons targeting PAXT and NEXT substrates were chosen from Figure 3B.
(E) UV-RIP assays from indicated HeLa LAP cells treated, or not, with cordycepin. Western blotting (left) and RT-qPCR (right) analyses were performed as in Figure 3C. For westerns, input samples represent 1% of protein extracts before IP. In right panel, cordycepin-treated samples are shown in lighter colors than their corresponding untreated samples.
All data are displayed as mean values with error bars denoting SDs (n = 3 biological replicates). Asterisks indicate detection of proteins of unknown origin.
See also Figure S5.
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 6 Models of NEXT- and PAXT-Dependent Nuclear RNA Decay
Schematic comparison of protein-protein links within the NEXT complex (left) and the PAXT connection (right). While both NEXT and PAXT pathways appear capable of detecting capped RNA by virtue of their physical linkages to the CBC, the different RNA-binding proteins (RBM7 for NEXT and PABPN1 for PAXT) discriminate their specificities. Question mark indicates that the ZFC3H1-PABPN1 linkage might not be direct. See text for details.
Molecular Cell  , DOI: ( /j.molcel )
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