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Defective structural RNA processing in relapsing-remitting multiple sclerosis.

Spurlock CF, Tossberg JT, Guo Y, Sriram S, Crooke PS, Aune TM - Genome Biol. (2015)

Bottom Line: We report profound defects in surveillance of structural RNAs in RRMS exemplified by elevated levels of poly(A) + Y1-RNA, poly(A) + 18S rRNA and 28S rRNAs, elevated levels of misprocessed 18S and 28S rRNAs and levels of the U-class of small nuclear RNAs.In cell lines, silencing of the genes encoding Ro60 and La proteins gives rise to these same defects in surveillance of structural RNAs.Our results establish that profound defects in structural RNA surveillance exist in RRMS and establish a causal link between Ro60 and La proteins and integrity of structural RNAs.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Vanderbilt University School of Medicine, Nashville, TN, 37232, USA. chase.spurlock@vanderbilt.edu.

ABSTRACT

Background: Surveillance of integrity of the basic elements of the cell including DNA, RNA, and proteins is a critical element of cellular physiology. Mechanisms of surveillance of DNA and protein integrity are well understood. Surveillance of structural RNAs making up the vast majority of RNA in a cell is less well understood. Here, we sought to explore integrity of processing of structural RNAs in relapsing remitting multiple sclerosis (RRMS) and other inflammatory diseases.

Results: We employed mononuclear cells obtained from subjects with RRMS and cell lines. We used quantitative-PCR and whole genome RNA sequencing to define defects in structural RNA surveillance and siRNAs to deplete target proteins. We report profound defects in surveillance of structural RNAs in RRMS exemplified by elevated levels of poly(A) + Y1-RNA, poly(A) + 18S rRNA and 28S rRNAs, elevated levels of misprocessed 18S and 28S rRNAs and levels of the U-class of small nuclear RNAs. Multiple sclerosis is also associated with genome-wide defects in mRNA splicing. Ro60 and La proteins, which exist in ribonucleoprotein particles and play different roles in quality control of structural RNAs, are also deficient in RRMS. In cell lines, silencing of the genes encoding Ro60 and La proteins gives rise to these same defects in surveillance of structural RNAs.

Conclusions: Our results establish that profound defects in structural RNA surveillance exist in RRMS and establish a causal link between Ro60 and La proteins and integrity of structural RNAs.

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Related in: MedlinePlus

Ro60 and La proteins are depressed in RRMS. (A) Ro60 (TROVE2) and La (SSB) transcript levels in CTRL (N = 24), CIS-MS (N = 16), RRMS (N = 22), RA (N = 18), SLE (N = 24), NMO (N = 22), and PD (N = 19) were determined by quantitative PCR after cDNA synthesis using oligo-dT. Results are normalized to CTRL = 1.0 after normalization to transcript levels of GAPDH, error bars are S.D. (B) As in (A) using whole genome RNA-sequencing data. (C) Western blotting to determine Ro60 and La protein levels in PBMC from CTRL (N = 9) and RRMS (N = 8). (D) Quantitative estimates of protein abundance relative to β-actin. *P <0.05, **P <0.01.
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Fig5: Ro60 and La proteins are depressed in RRMS. (A) Ro60 (TROVE2) and La (SSB) transcript levels in CTRL (N = 24), CIS-MS (N = 16), RRMS (N = 22), RA (N = 18), SLE (N = 24), NMO (N = 22), and PD (N = 19) were determined by quantitative PCR after cDNA synthesis using oligo-dT. Results are normalized to CTRL = 1.0 after normalization to transcript levels of GAPDH, error bars are S.D. (B) As in (A) using whole genome RNA-sequencing data. (C) Western blotting to determine Ro60 and La protein levels in PBMC from CTRL (N = 9) and RRMS (N = 8). (D) Quantitative estimates of protein abundance relative to β-actin. *P <0.05, **P <0.01.

Mentions: Ro60 and La proteins are components of ribonucleoprotein particles, bind discrete structural ncRNAs, and are thought to play important roles in ncRNA processing and quality control. For these reasons, we measured TROVE2 (Ro60) and SSB (La) expression levels in blood samples harvested in PaxGene tubes from the following cohorts of subjects: CTRL, CIS-MS, RRMS, RA, SLE, NMO, and PD. We found that TROVE2 and SSB transcript levels were markedly reduced in the established RRMS cohort compared to CTRL. This difference was unique to RRMS and not observed in other autoimmune disease cohorts or in other inflammatory (NMO) or non-inflammatory (PD) neurologic conditions (Figure 5A). We replicated these findings by whole-genome RNA sequencing (RNA-seq) and obtained equivalent results (Figure 5B). We also determined levels of protein expression of Ro60 and La in PBMC by western blotting. We found that both Ro60 and La proteins were profoundly diminished in RRMS PBMC relative to CTRL PBMC (Figure 5C and D). Thus, both TROVE2 and SSB transcripts and Ro60 and La proteins were profoundly diminished in RRMS and these mRNA and protein expression differences were not seen in several other autoimmune diseases.Figure 5


Defective structural RNA processing in relapsing-remitting multiple sclerosis.

Spurlock CF, Tossberg JT, Guo Y, Sriram S, Crooke PS, Aune TM - Genome Biol. (2015)

Ro60 and La proteins are depressed in RRMS. (A) Ro60 (TROVE2) and La (SSB) transcript levels in CTRL (N = 24), CIS-MS (N = 16), RRMS (N = 22), RA (N = 18), SLE (N = 24), NMO (N = 22), and PD (N = 19) were determined by quantitative PCR after cDNA synthesis using oligo-dT. Results are normalized to CTRL = 1.0 after normalization to transcript levels of GAPDH, error bars are S.D. (B) As in (A) using whole genome RNA-sequencing data. (C) Western blotting to determine Ro60 and La protein levels in PBMC from CTRL (N = 9) and RRMS (N = 8). (D) Quantitative estimates of protein abundance relative to β-actin. *P <0.05, **P <0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4403723&req=5

Fig5: Ro60 and La proteins are depressed in RRMS. (A) Ro60 (TROVE2) and La (SSB) transcript levels in CTRL (N = 24), CIS-MS (N = 16), RRMS (N = 22), RA (N = 18), SLE (N = 24), NMO (N = 22), and PD (N = 19) were determined by quantitative PCR after cDNA synthesis using oligo-dT. Results are normalized to CTRL = 1.0 after normalization to transcript levels of GAPDH, error bars are S.D. (B) As in (A) using whole genome RNA-sequencing data. (C) Western blotting to determine Ro60 and La protein levels in PBMC from CTRL (N = 9) and RRMS (N = 8). (D) Quantitative estimates of protein abundance relative to β-actin. *P <0.05, **P <0.01.
Mentions: Ro60 and La proteins are components of ribonucleoprotein particles, bind discrete structural ncRNAs, and are thought to play important roles in ncRNA processing and quality control. For these reasons, we measured TROVE2 (Ro60) and SSB (La) expression levels in blood samples harvested in PaxGene tubes from the following cohorts of subjects: CTRL, CIS-MS, RRMS, RA, SLE, NMO, and PD. We found that TROVE2 and SSB transcript levels were markedly reduced in the established RRMS cohort compared to CTRL. This difference was unique to RRMS and not observed in other autoimmune disease cohorts or in other inflammatory (NMO) or non-inflammatory (PD) neurologic conditions (Figure 5A). We replicated these findings by whole-genome RNA sequencing (RNA-seq) and obtained equivalent results (Figure 5B). We also determined levels of protein expression of Ro60 and La in PBMC by western blotting. We found that both Ro60 and La proteins were profoundly diminished in RRMS PBMC relative to CTRL PBMC (Figure 5C and D). Thus, both TROVE2 and SSB transcripts and Ro60 and La proteins were profoundly diminished in RRMS and these mRNA and protein expression differences were not seen in several other autoimmune diseases.Figure 5

Bottom Line: We report profound defects in surveillance of structural RNAs in RRMS exemplified by elevated levels of poly(A) + Y1-RNA, poly(A) + 18S rRNA and 28S rRNAs, elevated levels of misprocessed 18S and 28S rRNAs and levels of the U-class of small nuclear RNAs.In cell lines, silencing of the genes encoding Ro60 and La proteins gives rise to these same defects in surveillance of structural RNAs.Our results establish that profound defects in structural RNA surveillance exist in RRMS and establish a causal link between Ro60 and La proteins and integrity of structural RNAs.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Vanderbilt University School of Medicine, Nashville, TN, 37232, USA. chase.spurlock@vanderbilt.edu.

ABSTRACT

Background: Surveillance of integrity of the basic elements of the cell including DNA, RNA, and proteins is a critical element of cellular physiology. Mechanisms of surveillance of DNA and protein integrity are well understood. Surveillance of structural RNAs making up the vast majority of RNA in a cell is less well understood. Here, we sought to explore integrity of processing of structural RNAs in relapsing remitting multiple sclerosis (RRMS) and other inflammatory diseases.

Results: We employed mononuclear cells obtained from subjects with RRMS and cell lines. We used quantitative-PCR and whole genome RNA sequencing to define defects in structural RNA surveillance and siRNAs to deplete target proteins. We report profound defects in surveillance of structural RNAs in RRMS exemplified by elevated levels of poly(A) + Y1-RNA, poly(A) + 18S rRNA and 28S rRNAs, elevated levels of misprocessed 18S and 28S rRNAs and levels of the U-class of small nuclear RNAs. Multiple sclerosis is also associated with genome-wide defects in mRNA splicing. Ro60 and La proteins, which exist in ribonucleoprotein particles and play different roles in quality control of structural RNAs, are also deficient in RRMS. In cell lines, silencing of the genes encoding Ro60 and La proteins gives rise to these same defects in surveillance of structural RNAs.

Conclusions: Our results establish that profound defects in structural RNA surveillance exist in RRMS and establish a causal link between Ro60 and La proteins and integrity of structural RNAs.

Show MeSH
Related in: MedlinePlus