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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 polyadenylation and mis-processing of 18S and 28S rRNAs in RRMS. (A) Total rRNA levels in blood determined after cDNA synthesis using random hexamers in CTRL (N = 24) and RRMS (N = 22). (B) As in (A) except oligo-dT was used for cDNA synthesis. (C) Schematic illustrating PCR strategy to detect misprocessed 18S and 28S rRNAs. (D) Transcript levels of misprocessed rRNAs (-90, -60, -30) in RRMS (N = 12) relative to CTRL (N = 12). Results were normalized to total 18S or 28S RNA and are presented as fold difference between CTRL and RRMS. *P <0.05.
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Fig2: Increased polyadenylation and mis-processing of 18S and 28S rRNAs in RRMS. (A) Total rRNA levels in blood determined after cDNA synthesis using random hexamers in CTRL (N = 24) and RRMS (N = 22). (B) As in (A) except oligo-dT was used for cDNA synthesis. (C) Schematic illustrating PCR strategy to detect misprocessed 18S and 28S rRNAs. (D) Transcript levels of misprocessed rRNAs (-90, -60, -30) in RRMS (N = 12) relative to CTRL (N = 12). Results were normalized to total 18S or 28S RNA and are presented as fold difference between CTRL and RRMS. *P <0.05.

Mentions: We used a similar approach to measure levels of total and polyadenylated 18S and 28S rRNAs. We found that levels of total 18S and 28S rRNAs were similar between the RRMS and CTRL cohorts when random hexamers were used for cDNA synthesis (Figure 2A). However, we found a marked increase in levels of both polyadenylated 18S and 28S rRNAs as measured by employing oligo-dT for cDNA synthesis (Figure 2B). We therefore determined if there was also an increase in misprocessed rRNAs in RRMS since rRNAs are polyadenylated in the presence of defective processing of the 18S and 28S rRNAs from their 47S rRNA precursor. We predicted that misprocessing of rRNAs should produce 18S and 28S rRNAs longer than the typical 1.9 kb and 5.0 kb, respectively. To test this hypothesis, we designed a series of PCR primers to probe lengths of 18S and 28S rRNAs. We used common primers within the 18S or 28S rRNA (black arrows) and four additional primers; one within the classic 18S or 28S rRNA (green and blue arrows) and three that extend 30, 60, or 90 bp beyond the length of the fully processed 1.9 kb 18S or 5 kb 28S rRNA transcript (orange and red arrows) (Figure 2C). Random hexamers were used for cDNA synthesis. We found that 18S and 28S transcript levels in CTRL and RRMS were approximately the same (normalized to 1), labeled 18S (Figure 2D, left) or 28S (Figure 2D, right), when PCR primers within the 1.9 kb 18S rRNA or 5.0 kb 28S rRNA were employed. However, when we employed an internal primer (black arrow) and external primers 30, 60, or 90 bp distal to the normal transcript size (orange arrows, red arrows) we found a marked increase in transcript levels of 18S rRNA with extended ends between 30 and 90 bp in RRMS compared to CTRL. These 18S rRNA extended ends were more frequent at the 5’ end of the 18S rRNA than the 3’ end of the 18S rRNA. The 28S rRNA had only a modest increase in extended ends in RRMS relative to CTRL at the 5’ end. For the 28S RNA species, the increase in the frequency of extended ends in RRMS relative to CTRL was more pronounced at the 3’ end. We interpret these results to demonstrate that both 18S rRNAs and 28S rRNAs were not accurately processed to their mature size in RRMS.Figure 2


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 polyadenylation and mis-processing of 18S and 28S rRNAs in RRMS. (A) Total rRNA levels in blood determined after cDNA synthesis using random hexamers in CTRL (N = 24) and RRMS (N = 22). (B) As in (A) except oligo-dT was used for cDNA synthesis. (C) Schematic illustrating PCR strategy to detect misprocessed 18S and 28S rRNAs. (D) Transcript levels of misprocessed rRNAs (-90, -60, -30) in RRMS (N = 12) relative to CTRL (N = 12). Results were normalized to total 18S or 28S RNA and are presented as fold difference between CTRL and RRMS. *P <0.05.
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Related In: Results  -  Collection

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Fig2: Increased polyadenylation and mis-processing of 18S and 28S rRNAs in RRMS. (A) Total rRNA levels in blood determined after cDNA synthesis using random hexamers in CTRL (N = 24) and RRMS (N = 22). (B) As in (A) except oligo-dT was used for cDNA synthesis. (C) Schematic illustrating PCR strategy to detect misprocessed 18S and 28S rRNAs. (D) Transcript levels of misprocessed rRNAs (-90, -60, -30) in RRMS (N = 12) relative to CTRL (N = 12). Results were normalized to total 18S or 28S RNA and are presented as fold difference between CTRL and RRMS. *P <0.05.
Mentions: We used a similar approach to measure levels of total and polyadenylated 18S and 28S rRNAs. We found that levels of total 18S and 28S rRNAs were similar between the RRMS and CTRL cohorts when random hexamers were used for cDNA synthesis (Figure 2A). However, we found a marked increase in levels of both polyadenylated 18S and 28S rRNAs as measured by employing oligo-dT for cDNA synthesis (Figure 2B). We therefore determined if there was also an increase in misprocessed rRNAs in RRMS since rRNAs are polyadenylated in the presence of defective processing of the 18S and 28S rRNAs from their 47S rRNA precursor. We predicted that misprocessing of rRNAs should produce 18S and 28S rRNAs longer than the typical 1.9 kb and 5.0 kb, respectively. To test this hypothesis, we designed a series of PCR primers to probe lengths of 18S and 28S rRNAs. We used common primers within the 18S or 28S rRNA (black arrows) and four additional primers; one within the classic 18S or 28S rRNA (green and blue arrows) and three that extend 30, 60, or 90 bp beyond the length of the fully processed 1.9 kb 18S or 5 kb 28S rRNA transcript (orange and red arrows) (Figure 2C). Random hexamers were used for cDNA synthesis. We found that 18S and 28S transcript levels in CTRL and RRMS were approximately the same (normalized to 1), labeled 18S (Figure 2D, left) or 28S (Figure 2D, right), when PCR primers within the 1.9 kb 18S rRNA or 5.0 kb 28S rRNA were employed. However, when we employed an internal primer (black arrow) and external primers 30, 60, or 90 bp distal to the normal transcript size (orange arrows, red arrows) we found a marked increase in transcript levels of 18S rRNA with extended ends between 30 and 90 bp in RRMS compared to CTRL. These 18S rRNA extended ends were more frequent at the 5’ end of the 18S rRNA than the 3’ end of the 18S rRNA. The 28S rRNA had only a modest increase in extended ends in RRMS relative to CTRL at the 5’ end. For the 28S RNA species, the increase in the frequency of extended ends in RRMS relative to CTRL was more pronounced at the 3’ end. We interpret these results to demonstrate that both 18S rRNAs and 28S rRNAs were not accurately processed to their mature size in RRMS.Figure 2

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