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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

Loss of TROVE2 and/or SSB via small RNA interference increases poly(A) + Y1 RNA, rRNAs, and U1 RNA, misprocessed 18S and 28S rRNA, and alters mRNA length. (A) Selective siRNA-mediated knockdown of TROVE2 or SSB in THP-1 cells or Jurkat T cells. Transcript levels of TROVE2 and SSB were determined by quantitative PCR using oligo d(T) for cDNA synthesis. Results are normalized to cells transfected with a non-specific scrambled siRNA negative control after normalization to transcript levels of GAPDH. (B) Poly(A) + Y1 RNA levels were determined by quantitative PCR using oligo-dT for cDNA synthesis. Results normalized to CTRL (transfection of scrambled siRNA). (C) As in (B) except levels of 18S and 28S rRNAs were determined. (D) As in (B) except levels of poly(A) + U1 snRNA were determined. (E, F) Transcript levels of misprocessed rRNAs in THP-1 (E) and Jurkat T cells (F) (-90, -60, -30 bp) were determined and are expressed relative to cells transfected with a scrambled siRNA as described in Figure 2. (G) PCR strategy to test altered lengths of MBP, CSF1R, and NFATC1 transcripts. (H) As in (A) except transcript levels of the MBP intron, CSF1R exons, and NFATC1 short to long mRNA isoform ratios were determined by quantitative PCR and normalized to CTRL. Each experiment was performed a minimum of three times, results are expressed as mean ± S.D *P <0.05.
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Fig7: Loss of TROVE2 and/or SSB via small RNA interference increases poly(A) + Y1 RNA, rRNAs, and U1 RNA, misprocessed 18S and 28S rRNA, and alters mRNA length. (A) Selective siRNA-mediated knockdown of TROVE2 or SSB in THP-1 cells or Jurkat T cells. Transcript levels of TROVE2 and SSB were determined by quantitative PCR using oligo d(T) for cDNA synthesis. Results are normalized to cells transfected with a non-specific scrambled siRNA negative control after normalization to transcript levels of GAPDH. (B) Poly(A) + Y1 RNA levels were determined by quantitative PCR using oligo-dT for cDNA synthesis. Results normalized to CTRL (transfection of scrambled siRNA). (C) As in (B) except levels of 18S and 28S rRNAs were determined. (D) As in (B) except levels of poly(A) + U1 snRNA were determined. (E, F) Transcript levels of misprocessed rRNAs in THP-1 (E) and Jurkat T cells (F) (-90, -60, -30 bp) were determined and are expressed relative to cells transfected with a scrambled siRNA as described in Figure 2. (G) PCR strategy to test altered lengths of MBP, CSF1R, and NFATC1 transcripts. (H) As in (A) except transcript levels of the MBP intron, CSF1R exons, and NFATC1 short to long mRNA isoform ratios were determined by quantitative PCR and normalized to CTRL. Each experiment was performed a minimum of three times, results are expressed as mean ± S.D *P <0.05.

Mentions: To establish causal links between Ro60 and La imbalance and polyadenylation and processing of structural ncRNAs observed in RRMS, we designed siRNAs specific for human TROVE2 and SSB and transfected them into the human THP-1 monocyte or the Jurkat T cell line. Transfection with TROVE2 siRNA caused specific reduction of TROVE2 transcripts but not SSB transcripts (Figure 7A). Similarly, the SSB siRNA caused specific loss of SSB transcripts but not TROVE2 transcripts. We asked if reduced TROVE2 or SSB levels resulted in an increase in the amount of poly(A) + Y1 RNA in THP-1 cells. We found that knockdown of either TROVE2, SSB, or the combination effectively increased levels of poly(A) + Y1 RNA (Figure 7B). Additionally, knockdown of SSB resulted in marked accumulation of poly(A) + 18S rRNA (Figure 7C). Knockdown of TROVE2 resulted in only a modest increase in poly(A) + 18S RNA. In contrast, levels of poly(A) + 28S rRNA were largely unaffected by knockdown of either TROVE2 or SSB but were modestly increased by the knockdown of both TROVE2 and SSB. We also examined the impact of TROVE2 or SSB knockdown on levels of poly(A) + U1 RNA and found that knockdown of TROVE2, but not knockdown of SSB, caused a marked increase in levels of poly(A) + U1 RNA (Figure 7D). rRNA misprocessing was also analyzed as described above. In both THP-1 cells and Jurkat cells, transfection of TROVE2 and/or SBB siRNAs increased levels of misprocessed 18S rRNA and, to a lesser extent, misprocessed 28S rRNA (Figure 7E, F). Since the combination of TROVE2 and SSB knockdown increased the amount of poly(A) + U1 RNA and alterations in levels of U1 RNA result in isoform switching, we determined if TROVE2 and SSB RNA knockdown was sufficient to alter mRNA lengths in a cell [21]. We transfected TROVE2 and SSB siRNAs into THP-1 cells and employed a PCR strategy to test for different lengths of MBP, CSF1R, and NFATC1 transcripts (Figure 7G). Similar to what we observed in vivo, we found that reduced levels of either Ro60 or La by siRNA silencing were sufficient to increase expression levels of the MBP intron, to reduce expression of CSF1R exon 2 but not CSF1R exon 22, and also decrease the ratio of short to long NFATC1 isoforms (Figure 7H). Finally, we examined the ability of Ro60 and La to bind these target mRNAs using RNA immunoprecipitation and quantitative PCR. Only a modest fraction of total CSF1R, NFATC1, or MBP mRNA bound to Ro60 or La (Additional file 2: Figure S1). Taken together, these results demonstrate that Ro60 and La protein levels are critical for many aspects of proper RNA surveillance including maintaining low levels of the poly(A) + ncRNAs, Y1 RNA, rRNAs, and U RNA, rRNA processing, and proper mRNA splicing; key components of RNA function in the cell.Figure 7


Defective structural RNA processing in relapsing-remitting multiple sclerosis.

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

Loss of TROVE2 and/or SSB via small RNA interference increases poly(A) + Y1 RNA, rRNAs, and U1 RNA, misprocessed 18S and 28S rRNA, and alters mRNA length. (A) Selective siRNA-mediated knockdown of TROVE2 or SSB in THP-1 cells or Jurkat T cells. Transcript levels of TROVE2 and SSB were determined by quantitative PCR using oligo d(T) for cDNA synthesis. Results are normalized to cells transfected with a non-specific scrambled siRNA negative control after normalization to transcript levels of GAPDH. (B) Poly(A) + Y1 RNA levels were determined by quantitative PCR using oligo-dT for cDNA synthesis. Results normalized to CTRL (transfection of scrambled siRNA). (C) As in (B) except levels of 18S and 28S rRNAs were determined. (D) As in (B) except levels of poly(A) + U1 snRNA were determined. (E, F) Transcript levels of misprocessed rRNAs in THP-1 (E) and Jurkat T cells (F) (-90, -60, -30 bp) were determined and are expressed relative to cells transfected with a scrambled siRNA as described in Figure 2. (G) PCR strategy to test altered lengths of MBP, CSF1R, and NFATC1 transcripts. (H) As in (A) except transcript levels of the MBP intron, CSF1R exons, and NFATC1 short to long mRNA isoform ratios were determined by quantitative PCR and normalized to CTRL. Each experiment was performed a minimum of three times, results are expressed as mean ± S.D *P <0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Fig7: Loss of TROVE2 and/or SSB via small RNA interference increases poly(A) + Y1 RNA, rRNAs, and U1 RNA, misprocessed 18S and 28S rRNA, and alters mRNA length. (A) Selective siRNA-mediated knockdown of TROVE2 or SSB in THP-1 cells or Jurkat T cells. Transcript levels of TROVE2 and SSB were determined by quantitative PCR using oligo d(T) for cDNA synthesis. Results are normalized to cells transfected with a non-specific scrambled siRNA negative control after normalization to transcript levels of GAPDH. (B) Poly(A) + Y1 RNA levels were determined by quantitative PCR using oligo-dT for cDNA synthesis. Results normalized to CTRL (transfection of scrambled siRNA). (C) As in (B) except levels of 18S and 28S rRNAs were determined. (D) As in (B) except levels of poly(A) + U1 snRNA were determined. (E, F) Transcript levels of misprocessed rRNAs in THP-1 (E) and Jurkat T cells (F) (-90, -60, -30 bp) were determined and are expressed relative to cells transfected with a scrambled siRNA as described in Figure 2. (G) PCR strategy to test altered lengths of MBP, CSF1R, and NFATC1 transcripts. (H) As in (A) except transcript levels of the MBP intron, CSF1R exons, and NFATC1 short to long mRNA isoform ratios were determined by quantitative PCR and normalized to CTRL. Each experiment was performed a minimum of three times, results are expressed as mean ± S.D *P <0.05.
Mentions: To establish causal links between Ro60 and La imbalance and polyadenylation and processing of structural ncRNAs observed in RRMS, we designed siRNAs specific for human TROVE2 and SSB and transfected them into the human THP-1 monocyte or the Jurkat T cell line. Transfection with TROVE2 siRNA caused specific reduction of TROVE2 transcripts but not SSB transcripts (Figure 7A). Similarly, the SSB siRNA caused specific loss of SSB transcripts but not TROVE2 transcripts. We asked if reduced TROVE2 or SSB levels resulted in an increase in the amount of poly(A) + Y1 RNA in THP-1 cells. We found that knockdown of either TROVE2, SSB, or the combination effectively increased levels of poly(A) + Y1 RNA (Figure 7B). Additionally, knockdown of SSB resulted in marked accumulation of poly(A) + 18S rRNA (Figure 7C). Knockdown of TROVE2 resulted in only a modest increase in poly(A) + 18S RNA. In contrast, levels of poly(A) + 28S rRNA were largely unaffected by knockdown of either TROVE2 or SSB but were modestly increased by the knockdown of both TROVE2 and SSB. We also examined the impact of TROVE2 or SSB knockdown on levels of poly(A) + U1 RNA and found that knockdown of TROVE2, but not knockdown of SSB, caused a marked increase in levels of poly(A) + U1 RNA (Figure 7D). rRNA misprocessing was also analyzed as described above. In both THP-1 cells and Jurkat cells, transfection of TROVE2 and/or SBB siRNAs increased levels of misprocessed 18S rRNA and, to a lesser extent, misprocessed 28S rRNA (Figure 7E, F). Since the combination of TROVE2 and SSB knockdown increased the amount of poly(A) + U1 RNA and alterations in levels of U1 RNA result in isoform switching, we determined if TROVE2 and SSB RNA knockdown was sufficient to alter mRNA lengths in a cell [21]. We transfected TROVE2 and SSB siRNAs into THP-1 cells and employed a PCR strategy to test for different lengths of MBP, CSF1R, and NFATC1 transcripts (Figure 7G). Similar to what we observed in vivo, we found that reduced levels of either Ro60 or La by siRNA silencing were sufficient to increase expression levels of the MBP intron, to reduce expression of CSF1R exon 2 but not CSF1R exon 22, and also decrease the ratio of short to long NFATC1 isoforms (Figure 7H). Finally, we examined the ability of Ro60 and La to bind these target mRNAs using RNA immunoprecipitation and quantitative PCR. Only a modest fraction of total CSF1R, NFATC1, or MBP mRNA bound to Ro60 or La (Additional file 2: Figure S1). Taken together, these results demonstrate that Ro60 and La protein levels are critical for many aspects of proper RNA surveillance including maintaining low levels of the poly(A) + ncRNAs, Y1 RNA, rRNAs, and U RNA, rRNA processing, and proper mRNA splicing; key components of RNA function in the cell.Figure 7

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