1. Chromosomes and Gene Expression
  2. Microbiology and Infectious Disease
Download icon

Changes in mRNA abundance drive shuttling of RNA binding proteins, linking cytoplasmic RNA degradation to transcription

  1. Sarah Gilbertson
  2. Joel D Federspiel
  3. Ella Hartenian
  4. Ileana M Cristea
  5. Britt Glaunsinger  Is a corresponding author
  1. University of California, United States
  2. Princeton University, United States
  3. Howard Hughes Medical Institute, United States
Research Article
Cite this article as: eLife 2018;7:e37663 doi: 10.7554/eLife.37663
6 figures, 1 table, 1 data set and 2 additional files

Figures

Figure 1 with 1 supplement
RNA binding proteins are translocated from the cytoplasm to the nucleus in cells undergoing enhanced cytoplasmic mRNA decay.

(A) Diagram depicting the experimental setup. (B) Venn diagram of nuclear proteins that are specifically and significantly (p<0.05) enriched in muSOX-expressing cells compared to D219A-expressing cells that also show either no change or a decrease in cytoplasmic abundance. (C) Gene ontology molecular function overrepresentation analysis by Pantherdb, graphed according to their P value. (D) Graphs showing the nuclear and cytoplasmic distribution of poly(A) binding proteins from the TMT-MS data. Graphs display the mean with SEM of 3 biological replicates. (E) Western blot of nuclear, cytoplasmic, and whole cell fractions of NIH3T3 fibroblasts mock infected or infected with WT or R443I MHV68 for 24 hr. GAPDH and histone H3 serve as fractionation and loading controls. Shown is a representative example of 3 biological replicates.

https://doi.org/10.7554/eLife.37663.003
Figure 1—figure supplement 1
RNA binding proteins are translocated from the cytoplasm to the nucleus in cells undergoing enhanced mRNA decay.

(A) Western blots of nuclear, cytoplasmic, and whole cell fractions of HEK293T cells transfected with either empty vector control, WT muSOX, or D219A muSOX. Shown are two independent biological replicates. GAPDH and histone H3 serve as fractionation and loading controls. (B, C) Graphs showing poly(U) (B) and 3' UTR (C) binding protein distributions in the nucleus and cytoplasm from the TMT-LC/MS-MS data. Graphs display the mean with SEM of 3 biological replicates.

https://doi.org/10.7554/eLife.37663.004
Figure 2 with 1 supplement
Nuclear translocation of RNA binding proteins is dependent on mRNA degradation by Xrn1.

(A) Pie chart showing the percent of shuttling proteins that fail to translocate in Xrn1 KO cells. (B) Heat map depicting the average nuclear abundance in WT or Xrn1 KO HEK293T cells of the 67 significantly shifted proteins in samples expressing muSOX or D219A, relative to the empty vector control. (C) Western blots of nuclear, cytoplasmic, and whole cell fractions of WT (left panel) or Xrn1 KO (right panel) HEK293T cells transfected with the indicated plasmid. GAPDH and histone H3 serve as fractionation and loading controls. (D) Confocal microscopy and quantification of WT or Xrn1 KO HEK293T cells transfected with GFP or GFP-muSOX, showing signals for DAPI stained nuclei (blue), PABPC (red), GFP (green), and the merged images (overlay). Arrow heads point to representative GFP-muSOX expressing cells. The number of cells displaying either cytoplasmic or nuclear PABPC localization by immunofluorescence (IFA) was quantified for WT or Xrn1 KO cells expressing GFP-muSOX. A total of 75 GFP-muSOX WT cells and 80 GFP-muSOX Xrn1 KO cells were counted. The graph displays individual biological replicates as dots, with the mean and SEM.

https://doi.org/10.7554/eLife.37663.005
Figure 2—figure supplement 1
Nuclear translocation of RNA binding proteins is dependent on mRNA degradation by Xrn1.

(A) Western blots showing the levels of Xrn1 or the GAPDH loading control in WT or Xrn1 KO HEK293T cells. (B) WT or Xrn1 KO HEK293T cells transfected with either empty vector or muSOX were subjected to ChIP using antibodies to RNAPII or IgG. Purified chromatin was quantified by qPCR. Graphs display individual biological replicates as dots, with the mean and SEM. Statistical significance was determined using Student’s t test *p<0.05 **p<0.005 ***p<0.0005. (C) Growth curve of WT or Xrn1 KO HEK293T cells. Statistical significance was determined using Student’s t test *p<0.05 **p<0.005 ***p<0.0005. (D) Confocal microscopy of WT or Xrn1 KO HEK293T cells transfected with GFP or GFP-muSOX, showing signals for DAPI stained nuclei (blue), LARP4 (red), GFP (green), and the merged images (overlay). Arrow heads point to representative GFP-muSOX expressing cells.

https://doi.org/10.7554/eLife.37663.006
Figure 3 with 1 supplement
Xrn1 knockout leads to subcellular redistribution of proteins functionally associated with RNA.

(A) The number of proteins that are differentially expressed in Xrn1 knockout (KO) cells from the nucleus (149) and the cytoplasm (158). Gene ontology molecular function overrepresentation analysis by Pantherdb is shown for each compartment, graphed according to their false discovery rate (FDR). (B) Graphs showing the nuclear and cytoplasmic distribution of decapping-related proteins from the TMT-LC/MS-MS data. Graphs display the mean with SEM of 3 biological replicates. (C) Heatmap depicting the Log2 abundance ratio in Xrn1 KO HEK293T cells compared to WT HEK293T cells of proteins identified as Xrn1 interactors using the BioGRID database. Proteins with a significant difference in abundance between WT and Xrn1 KO are listed in red. (D) Graph of nuclear and cytoplasmic distribution of GW182 from the TMT-LC/MS-MS data. Graph displays the mean with SEM of 3 biological replicates. (E) Western blot of nuclear, cytoplasmic, and whole cell fractions of WT and Xrn1 KO HEK293T cells. GAPDH and histone H3 serve as fractionation and loading controls.

https://doi.org/10.7554/eLife.37663.007
Figure 3—figure supplement 1
Xrn1 knockout leads to subcellular redistribution of proteins functionally associated with RNA.

(A) Graphs showing the distribution of proteins of the oligosaccharyltransferase (OST) complex in the nucleus and cytoplasm from the TMT-LC/MS-MS data. Graphs display the mean with SEM of 3 biological replicates. (B) mRNA levels from WT and Xrn1 KO HEK293T cells were measured by RT-qPCR. Graphs display individual biological replicates as dots, with the mean and SEM. Statistical significance was determined using Student’s t test *p<0.05 **p<0.005 ***p<0.0005.

https://doi.org/10.7554/eLife.37663.008
LARP4 translocates to the nucleus in a PABPC-dependent manner.

(A) STRING network of reported protein-protein interactions between the 67 proteins that shuttle in muSOX-expressing cells. Medium and high confidence interactions are shown with thin and thick connector lines, respectively. (B, C) Western blots of nuclear and cytoplasmic fractions of vector- or muSOX-transfected HEK293T cells treated with the indicated siRNA. GAPDH and histone H3 serve as fractionation and loading controls.

https://doi.org/10.7554/eLife.37663.009
Figure 5 with 2 supplements
PABPC depletion prevents muSOX-induced repression of RNAPII recruitment.

(A, B) HEK293T cells treated with siRNAs targeting PABPC1 and 4 (A), LARP4 (B), or non-targeting scramble siRNAs were subsequently transfected with either empty vector or muSOX, then subjected to chromatin immunoprecipitation (ChIP) using antibodies to RNAPII or IgG. Western blots showing protein levels of PABPC1, PABPC4, and Larp4 after siRNA depletion are shown in the lower panels, along with a GAPDH loading control. (C) Western blots of nuclear and cytoplasmic fractions of HEK293T cells transfected with an empty vector or a plasmid containing FLAG-PABPC1. GAPDH and histone H3 serve as fractionation and loading controls. (D) HEK293T cells transfected with either empty vector or FLAG-PABPC1 were subjected to ChIP using antibodies to RNAPII or IgG. (E) WT or Xrn1 KO HEK293T cells transfected with either empty vector or FLAG-PABPC1 alone or together with muSOX were subjected to ChIP using antibodies to RNAPII or IgG. Purified chromatin in each of the above experiments was quantified by qPCR. Western blots showing the levels of Xrn1 in WT or Xrn1KO HEK293Ts are shown, along with a GAPDH loading control. All graphs display individual biological replicates as dots, with the mean and SEM. Statistical significance was determined using Student’s t test *p<0.05 **p<0.005 ***p<0.0005.

https://doi.org/10.7554/eLife.37663.010
Figure 5—figure supplement 1
PABPC is required for the connection between cytoplasmic mRNA decay and RNAPII promoter occupancy.

(A,C) HEK293T cells were treated with siRNAs targeting CHD3 (A), MSI1 (B), or TRIM32 (C), or non-targeting scramble siRNAs, then transfected with either empty vector or muSOX and subjected to chromatin immunoprecipitation (ChIP) using antibodies to RNAPII or IgG. Purified chromatin was quantified by qPCR. Western blots showing the levels of CHD3 and MSI1 after siRNA depletion, along with a GAPDH or histone H3 loading control are shown below. Graphs display individual biological replicates as dots, with the mean and SEM. Statistical significance was determined using Student’s t test *p<0.05 **p<0.005 ***p<0.0005.

https://doi.org/10.7554/eLife.37663.011
Figure 5—figure supplement 2
Effects of depleting PABPC, LARP4, CHD3, MSI1 and TRIM32 on RNAPII promoter occupancy.

(A–E) HEK293T cells were treated with siRNAs targeting PABPC1 and PABPC4 (A), LARP4 (B), CHD3 (C), MSI1 (D), or TRIM32 (E), and subjected to ChIP using antibodies to RNAPII or IgG. Purified chromatin was quantified by qPCR. All graphs display individual biological replicates as dots, with the mean and SEM. Statistical significance was determined using Student’s t test *p<0.05 **p<0.005 ***p<0.0005.

https://doi.org/10.7554/eLife.37663.012
Figure 6 with 1 supplement
Nuclear translocation of PABPC selectively impacts early stages of transcription.

(A) HEK293T cells transfected with empty vector, muSOX, or FLAG-PABPC1 were subjected to ChIP using antibodies to RNAPII or IgG at the indicated gene matched promoters and exons. (B) ChIP using antibodies to serine 5-phosphorylated (Ser5P) RNAPII or IgG at gene promoters. The level of Ser5P RNAPII was determined by dividing the Ser5P values over the total RNAPII values within the same region of the gene in HEK293T cells transfected with empty vector, muSOX, or FLAG-PABPC1. (C) ChIP was performed as described in (B), but using antibodies to serine 2-phosphorylated (Ser2P) RNAPII or IgG at gene exons. The level of Ser2P RNAPII was determined by dividing the Ser2P values over the total RNAPII values within the same region of the gene. (D) ChIP was performed as described in (B), but using antibodies to TATA-binding protein (TBP) or IgG at gene promoters. (E) ChIP was performed as described in (B), but using antibodies to the POLR3A subunit of RNAPIII or IgG. In each experiment, chromatin was quantified by qPCR and all graphs display individual biological replicates as dots, with the mean and SEM. Statistical significance was determined using Student’s t test *p<0.05 **p<0.005 ***p<0.0005. (F) Model summarizing the impact of SOX and Xrn1-driven mRNA degradation on RBP trafficking and RNAPII transcription. See text for details.

https://doi.org/10.7554/eLife.37663.013
Figure 6—figure supplement 1
RNAPII Rpb1 and TBP protein levels are unchanged in cells expressing muSOX or FLAG-PABPC1.

Western blot of cells expressing empty vector, muSOX, or FLAG-PABPC1, with VINCULIN serving as a loading control.

https://doi.org/10.7554/eLife.37663.014

Tables

Key resources table
Reagent type
(species) or resource
DesignationSource or referenceIdentifiersAdditional
information
Strain, strain
background
(murine herpesvirus 68)
MHV68PMID: 10888635NCBI_refseq ID:
NC_001826.2
Koszinowski
Lab
Strain,
strain background
(murine herpesvirus 68)
R443I MHV68PMID: 21811408Glaunsinger
Lab
Cell line
(Homo sapiens)
HEK293TAmerican Type
Culture Collection
Cat# CRL-11268;
RRID: CVCL_1926
Cell line
(Mus musculus)
NIH3T3American Type
Culture Collection
Cat# CRL-1658;
RRID: CVCL_0594
Transfected
construct (synthesized)
Xrn1 knockout
(KO) cells
This paperHEK293T clone
stably expressing Cas9
and Xrn1 single-guide RNA.
Transfected
construct (synthesized)
Cas9-expressing
WT cells
This paperHEK293T clone
stably expressing
Cas9 alone with
no guide RNA.
Recombinant DNA
reagent
GFP-muSOX
(plasmid)
This paperProgenitors:
pcDNA3-HA-muSOX (
Covarrubias et al., 2009);
Gateway vector peGFP-C1
Recombinant D
NA reagent
Thy1.1-muSOX
(plasmid)
This paperProgenitors:
GFP-muSOX
Recombinant
DNA reagent
Thy1.1-muSOX D219A
(plasmid)
This paperProgenitors:
Thy1.1-muSOX
Recombinant
DNA reagent
Thy1.1-GFP (plasmid)This paperProgenitors:
Thy1.1-muSOX
Recombinant
DNA reagent
pCDEF3-FLAG-PABPC1
(plasmid)
PMID: 20823266Glaunsinger Lab
Recombinant
DNA reagent
lentiCas9-Blast
(lentiviral vector)
Addgene;
PMID: 25075903;
PMID: 24336571
52962
Recombinant
DNA reagent
lentiGuide-Puro
(lentiviral vector)
Addgene;
PMID: 25075903;
PMID: 24336571
52963
AntibodyMouse monoclonal
anti-PABPC
Santa Cruz
Biotechnologies
Clone 10 E 10; SC32318IFA (1:25)
AntibodyRabbit polyclonal
anti-LARP4
ThermofisherPA5-58727IFA (1:200);
Western (1:1000)
AntibodyRabbit polyclonal
anti-PABPC
Cell Signaling
Technology
4992SWestern (1:1000)
AntibodyRabbit polyclonal
anti-PABPC4
BethylA301-466A-MWestern (1:1000)
AntibodyMouse monoclonal
anti-Gapdh
AbcamClone 6C5; ab8245Western (1:5000)
AntibodyRabbit monoclonal
anti-Histone H3
Cell Signaling
Technology
Clone D1H2; 4499SWestern (1:2000)
AntibodyRabbit polyclonal
anti-LYRIC
Abcamab124789Western (1:1000)
AntibodyRabbit polyclonal
anti-RRBP1
BethylA303-996A-TWestern (1:1000)
AntibodyRabbit polyclonal
anti-MSI1
Abcamab52865Western (1:1000)
AntibodyRabbit polyclonal
anti-LIN28B
Abcamab71415Western (1:1000)
AntibodyRabbit polyclonal
anti-RPP20
Novus BiologicalsNBP15707220Western (1:1000)
AntibodyRabbit polyclonal
anti-CHD3
Cell Signaling
Technology
4241TWestern (1:1000)
AntibodyRabbit polyclonal
anti-THOC6
Life TechnologiesPA543172Western (1:1000)
AntibodyRabbit polyclonal
anti-PNN
Life TechnologiesPA535053Western (1:1000)
AntibodyRabbit polyclonal
anti-EXO4
This paperWestern (1:1000)
AntibodyRabbit polyclonal
anti-NPM
Abcamab10530Western (1:1000)
AntibodyRabbit polyclonal
anti-TRIM32
Abcamab131223Western (1:1000)
AntibodyMouse monoclonal
anti-GW182
AbcamClone 4B6; ab70522Western (1:1000)
AntibodyRabbit polyclonal
anti-DDX6
BethylA300-460Western (1:1000)
AntibodyRabbit polyclonal
anti-DCP2
BethylA302-597A-MWestern (1:1000)
AntibodyMouse monoclonal
anti-RNAPII Rpb1
BioLegend8WG16Western
(1:2000); ChIP (10 mg)
AntibodyRabbit polyclonal
anti-TBP
Abcamab28175Western
(1:2000); ChIP (10 mg)
AntibodyRabbit polyclonal
anti-RNAPII
phospho S5
Abcamab5131ChIP (10 mg)
AntibodyRabbit polyclonal
anti-RNAPII
phospho S2
Abcamab5095ChIP (10 mg)
AntibodyRabbit polyclonal
anti-POLR3A
Abcamab96328ChIP (10 mg)
Sequence-based
reagent (H. sapiens)
siPABPC1PMID: 20823266Custom siRNA;
see Table S5 for
sequence
Sequence-based
reagent (H. sapiens)
siPABPC4PMID: 20823266Custom siRNA;
see Table S5 for
sequence
Sequence-based
reagent (H. sapiens)
siLARP4DharmaconM-016523-00-0020
Sequence-based
reagent (H. sapiens)
siCHD3DharmaconM-023015-01-0020
Sequence-based
reagent (H. sapiens)
siMSI1DharmaconM-011338-01-0010
Sequence-based
reagent (H. sapiens)
siTRIM32DharmaconM-006950-01-0010
Commercial assay or kitProtein G dynabeadsThermofisher10004D
Commercial assay or kitProtein A dynabeadsThermofisher10002D
Commercial assay or kit10-plex TMT kitThermofisher90113
Chemical compound, drugAlexa Fluor 594,
goat anti-rabbit
ThermofisherA-11072
Chemical compound, drugAlexa Fluor 594,
goat anti-mouse
ThermofisherA-11020
Software, algorithmProteome
Discoverer
Thermofisherv2.2.0.388

Data availability

Mass spectrometry proteomics data reported in this paper have been deposited at the ProteomeXchange Consortium via the PRIDE partner repository under accession number PXD009487.

The following data sets were generated
  1. 1

Additional files

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Download citations (links to download the citations from this article in formats compatible with various reference manager tools)

Open citations (links to open the citations from this article in various online reference manager services)