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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

Increased total and polyadenylated U snRNAs in RRMS. (A) Total U1 RNA transcript levels were determined by quantitative PCR using random hexamers for cDNA synthesis in CTRL (N = 24) and RRMS (N = 22). Individual transcripts were normalized to GAPDH transcript levels. (B) As in (A) except total poly(A) + U1 snRNA levels were determined by quantitative PCR using oligo-dT for cDNA synthesis in CTRL (N = 24), CIS-MS (N = 16), RRMS (N = 22), SLE (N = 24), RA (N = 18), and NMO (N = 22). (C) As in (A), except transcript levels were determined by whole genome RNA-sequencing (RNA-seq) and normalized to CTRL = 1; CTRL (N = 8) and RRMS (N = 6). (D) As in (A, B) except transcript levels of U11 and U12 snRNAs were determined by quantitative PCR using oligo d(T) for poly(A) + or random hexamer primers for total U11 or U12 snRNA.
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Fig3: Increased total and polyadenylated U snRNAs in RRMS. (A) Total U1 RNA transcript levels were determined by quantitative PCR using random hexamers for cDNA synthesis in CTRL (N = 24) and RRMS (N = 22). Individual transcripts were normalized to GAPDH transcript levels. (B) As in (A) except total poly(A) + U1 snRNA levels were determined by quantitative PCR using oligo-dT for cDNA synthesis in CTRL (N = 24), CIS-MS (N = 16), RRMS (N = 22), SLE (N = 24), RA (N = 18), and NMO (N = 22). (C) As in (A), except transcript levels were determined by whole genome RNA-sequencing (RNA-seq) and normalized to CTRL = 1; CTRL (N = 8) and RRMS (N = 6). (D) As in (A, B) except transcript levels of U11 and U12 snRNAs were determined by quantitative PCR using oligo d(T) for poly(A) + or random hexamer primers for total U11 or U12 snRNA.

Mentions: Next, we determined levels of U1 RNA in blood (PaxGene tubes) from CTRL subjects, subjects with RRMS or with other inflammatory diseases. As above, synthesis of cDNA was performed using random hexamers and oligo-dT and transcript levels were determined by quantitative PCR. We found a modest increase in total levels of U1 RNA (random hexamers) in RRMS relative to CTRL (Figure 3A). In contrast, poly(A) + U1 transcript levels were markedly elevated in subjects with RRMS relative to CTRL (Figure 3B). The CIS-MS cohort did not exhibit increased poly(A) + U1 RNA levels nor did subjects with other inflammatory diseases, SLE, RA, and NMO. We also extracted RNA-seq data and confirmed that poly(A) + U1 RNA levels were elevated in RRMS and that U2 and U4 RNA levels were also elevated in RRMS relative to CTRL (Figure 3C). We also examined expression of total and poly(A) + U11 and U12 transcripts, components of the minor spliceosome pathway, and found elevated poly(A) + U11 and U12 transcript levels in RRMS versus CTRL and increased total U11 transcript levels. Total levels of U12 snRNA were not significantly different between RRMS and CTRL (Figure 3D). Thus, multiple ncRNA species exhibit increased polyadenylation in RRMS relative to CTRL or other autoimmune diseases.Figure 3


Defective structural RNA processing in relapsing-remitting multiple sclerosis.

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

Increased total and polyadenylated U snRNAs in RRMS. (A) Total U1 RNA transcript levels were determined by quantitative PCR using random hexamers for cDNA synthesis in CTRL (N = 24) and RRMS (N = 22). Individual transcripts were normalized to GAPDH transcript levels. (B) As in (A) except total poly(A) + U1 snRNA levels were determined by quantitative PCR using oligo-dT for cDNA synthesis in CTRL (N = 24), CIS-MS (N = 16), RRMS (N = 22), SLE (N = 24), RA (N = 18), and NMO (N = 22). (C) As in (A), except transcript levels were determined by whole genome RNA-sequencing (RNA-seq) and normalized to CTRL = 1; CTRL (N = 8) and RRMS (N = 6). (D) As in (A, B) except transcript levels of U11 and U12 snRNAs were determined by quantitative PCR using oligo d(T) for poly(A) + or random hexamer primers for total U11 or U12 snRNA.
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Related In: Results  -  Collection

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Fig3: Increased total and polyadenylated U snRNAs in RRMS. (A) Total U1 RNA transcript levels were determined by quantitative PCR using random hexamers for cDNA synthesis in CTRL (N = 24) and RRMS (N = 22). Individual transcripts were normalized to GAPDH transcript levels. (B) As in (A) except total poly(A) + U1 snRNA levels were determined by quantitative PCR using oligo-dT for cDNA synthesis in CTRL (N = 24), CIS-MS (N = 16), RRMS (N = 22), SLE (N = 24), RA (N = 18), and NMO (N = 22). (C) As in (A), except transcript levels were determined by whole genome RNA-sequencing (RNA-seq) and normalized to CTRL = 1; CTRL (N = 8) and RRMS (N = 6). (D) As in (A, B) except transcript levels of U11 and U12 snRNAs were determined by quantitative PCR using oligo d(T) for poly(A) + or random hexamer primers for total U11 or U12 snRNA.
Mentions: Next, we determined levels of U1 RNA in blood (PaxGene tubes) from CTRL subjects, subjects with RRMS or with other inflammatory diseases. As above, synthesis of cDNA was performed using random hexamers and oligo-dT and transcript levels were determined by quantitative PCR. We found a modest increase in total levels of U1 RNA (random hexamers) in RRMS relative to CTRL (Figure 3A). In contrast, poly(A) + U1 transcript levels were markedly elevated in subjects with RRMS relative to CTRL (Figure 3B). The CIS-MS cohort did not exhibit increased poly(A) + U1 RNA levels nor did subjects with other inflammatory diseases, SLE, RA, and NMO. We also extracted RNA-seq data and confirmed that poly(A) + U1 RNA levels were elevated in RRMS and that U2 and U4 RNA levels were also elevated in RRMS relative to CTRL (Figure 3C). We also examined expression of total and poly(A) + U11 and U12 transcripts, components of the minor spliceosome pathway, and found elevated poly(A) + U11 and U12 transcript levels in RRMS versus CTRL and increased total U11 transcript levels. Total levels of U12 snRNA were not significantly different between RRMS and CTRL (Figure 3D). Thus, multiple ncRNA species exhibit increased polyadenylation in RRMS relative to CTRL or other autoimmune diseases.Figure 3

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