RNF130 Regulates LDLR Availability and Plasma LDL Cholesterol Levels

Pcsk9-/- mice are a model of increased hepatic LDL receptors.

“We overexpressed RNF130 in Pcsk9–/– mice, a model of increased hepatic LDL receptors27,28.”

• cites [27] Tavori H, Fan D, Blakemore JL, Yancey PG, Ding L, Linton S, Fazio S. Serum proprotein convertase subtilisin/kexin type 9 and cell surface low-density lipoprotein receptor: evidence for a reciprocal regulation. 2014; 127: 2403– 2413. PMC3806304 23690465 sourcePartly supported

  The Results section on PCSK9 and LDLR expression reports that expressing human PCSK9 reduced hepatic LDLR levels by 41%, supporting the underlying relationship. But the paper studies PCSK9-overexpressing mice, not Pcsk9−/− mice, and does not establish that the knockout mice are a model of increased hepatic LDLR. (LLM, conf=0.81)

  Experimental — accuracy not yet measured
• cites [28] Lagace TA, Curtis DE, Garuti R, McNutt MC, Sahng WP, Prather HB, Anderson NN, Ho YK, Hammer RE, Horton JD. Secreted PCSK9 decreases the number of LDL receptors in hepatocytes and in livers of parabiotic mice. J Clin Invest. 2006; 116: 2995– 3005. 17080197 PMC1626117 sourceSupported

Loss-of-function mutations of the RNF130 gene are significantly lower than the expected odds in publicly available human data.

“This is in agreement with publicly available human data, in which loss-of-function mutations of the RNF130 gene are significantly lower than the expected odds. Only nine loss-of-function (LoF) mutations in RNF130 have been described in the gnomAD database, which is 46% of the expected rate of LoF mutation45.”

• cites [45] Karczewski KJ, Francioli LC, Tiao G, Cummings BB, Alföldi J, Wang Q, Collins RL, Laricchia KM, Ganna A, Birnbaum DP, The mutational constraint spectrum quantified from variation in 141,456 humans. bioRxiv. 2020; 531210. PMC7334197 32461654 sourceCould not verify

  The Abstract identifies this paper as describing gnomAD, and ‘The LoF intolerance of human genes’ explains its gene-level observed/expected metric. However, our copy does not report an RNF130 result or the claimed nine variants and 46% figure (searched: RNF130 and those numbers). (LLM, conf=0.72)

  Experimental — accuracy not yet measured

Currently no homozygotes have been identified for these RNF130 loss-of-function mutations.

“Additionally, currently no homozygotes have been identified for these mutations, suggesting that loss of RNF130 is not well tolerated.”

• cites [45] Karczewski KJ, Francioli LC, Tiao G, Cummings BB, Alföldi J, Wang Q, Collins RL, Laricchia KM, Ganna A, Birnbaum DP, The mutational constraint spectrum quantified from variation in 141,456 humans. bioRxiv. 2020; 531210. PMC7334197 32461654 sourceCould not verify

  The ‘Identifying loss-of-function variants’ section reports homozygous predicted loss-of-function variants across the dataset, but does not give a result for RNF130. Searches for RNF130 and for homozygous RNF130 loss-of-function mutations found no passage establishing whether homozygotes were identified for the mutations in question. (LLM, conf=0.76)

  Experimental — accuracy not yet measured

The 50% decrease in LDL-C with ASO silencing of Rnf130 is comparable to inhibiting PCSK9.

“ASO silencing of Rnf130 resulted in a 50% decrease in LDL-C, a change comparable to inhibiting PCSK940,41, further supporting that RNF130 is a significant regulator of LDLR and LDL-C levels.”

• cites [40] Stein EA, Mellis S, Yancopoulos GD, Stahl N, Logan D, Smith WB, Lisbon E, Gutierrez M, Webb C, Wu R, Effect of a monoclonal antibody to PCSK9 on LDL cholesterol. Obstet Gynecol Surv. 2012; 67: 413– 414. 22435370Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

• cites [41] Blom DJ, Hala T, Bolognese M, Lillestol MJ, Toth PD, Burgess L, Ceska R, Roth E, Koren MJ, Ballantyne CM, A 52-week placebo-controlled trial of evolocumab in hyperlipidemia. N Engl J Med. 2014; 370: 1809– 1819. 24678979 sourceSupported

A recent CRISPR screen identified disruption of both RNF130 and IDOL as upregulating cell surface levels of LDLR.

“Of note, this latter study identified disruption of both RNF130 and IDOL as upregulating cell surface levels of LDLR, consistent with RNF130 functioning to regulate LDLR protein levels at the cell surface.”

• cites [37] Trinh MN, Brown MS, Goldstein JL, Han J, Vale G, McDonald JG, Seemann J, Mendell JT, Lu F. Last step in the path of LDL cholesterol from lysosome to plasma membrane to ER is governed by phosphatidylserine. Proc Natl Acad Sci U S A. 2020; 117: 18521– 18529. 32690708 PMC7414171 sourcePartly supported

  The Results section describes a CRISPR screen that selected cells with high surface LDLR antibody binding. It explicitly says IDOL was the second-ranked gene and that cells lacking IDOL scored positive because they have high LDLR levels. Our copy does not identify RNF130 as a hit, so it does not support the claim about both genes. (LLM, conf=0.86)

  Experimental — accuracy not yet measured

FLAG-RNF130 overexpression in WT mice increased LDL cholesterol levels.

“FLAG-RNF130 overexpression in WT mice (Fig. S3A–B) increased total and LDL cholesterol levels (Fig. 3B–C)”

• AssumptionRests on an assumption: Human RNF130 functions equivalently to mouse RNF130 in regulating LDLR and plasma LDL-C.

Cells expressing mutant RNF130C304A continued to express high levels of LDLR both at the cell surface and in intracellular compartments.

“cells expressing mutant RNF130C304A continued to express high levels of LDLR both at the cell surface and in intracellular compartments (Fig. 2E; right panel and Fig. 2F).”

The reduction in plasma cholesterol in Rnf130-/- mice appeared to be present in the LDL fraction.

“Nevertheless, importantly plasma lipid analysis in Rnf130–/– mice showed a significant reduction in circulating plasma total cholesterol (Fig. 5C) and these changes appeared to be present in the LDL fraction (Fig. 5D).”

RNF130 ubiquitinates LDLR.

“we have determined that RNF130 ubiquitinates the LDLR”

• Also stated in: § Results: · boosted

  “We demonstrate that RNF130 is an E3 ubiquitin ligase that ubiquitinates LDLR”

Overexpression of RNF130 in Pcsk9-/- mice did not change plasma TAG levels.

“Overexpression of RNF130 in Pcsk9–/– mice (Fig. 4A–B) resulted in increased plasma total cholesterol (Fig. 4C, 4E), LDL/VLDL-C (Fig. 4D–E), and HDL-C levels (Fig. S4A), with no change in plasma TAG levels (Fig. S4B).”

There was no significant difference in body weight between animals treated with control AAV-CRISPR or Rnf130 AAV-CRISPR after two weeks.

“After two weeks, there was no significant difference in body weight between animals treated with either control AAV-CRISPR or Rnf130 AAV-CRISPR (Fig. S6C).”

Combined mutation of all four residues (K811/816/830R/C839A) prevented ubiquitination of LDLR.

“Combined mutation of all four residues (K811/816/830R/C839A) prevented both ubiquitination of LDLR (Fig. 2B; Ubiquitination)”

Overexpression of RNF130 in Ldlr-/- mice did not change plasma total cholesterol.

“Overexpression of RNF130 (Fig. S3D) in Ldlr–/– mice did not change plasma total cholesterol (Fig. 3E, F)”

• AssumptionRests on an assumption: Human RNF130 functions equivalently to mouse RNF130 in regulating LDLR and plasma LDL-C.

RNF130 overexpression resulted in significantly increased non-HDL (LDL/VLDL) plasma cholesterol levels.

“RNF130 overexpression resulted in significantly increased total (Fig. 1G) and non-HDL (LDL/VLDL) plasma cholesterol levels (Fig. 1H).”

• AssumptionRests on an assumption: Human RNF130 functions equivalently to mouse RNF130 in regulating LDLR and plasma LDL-C.

Overexpression of RNF130 in Pcsk9-/- mice increased LDL/VLDL-C levels by 70%.

“Together these data show that overexpression of RNF130 in WT and Pcsk9–/– mice increased LDL/VLDL-C levels by 36% (Fig. 1H–I) and 70% (Fig. 4D–E), respectively, suggesting that the efficacy of RNF130 for increasing LDL/VLDL-C is enhanced when basal hepatic LDLR levels are elevated.”

There was no change in intracellular LDLR levels following co-expression of WT RNF130.

“Analysis of intracellular LDLR levels indicated that there was no change in intracellular LDLR levels following co-expression of WT RNF130 (Fig. 2D; IB:GFP lane 11 vs. 10).”

Mutant RNF130C304A did not reduce cell surface GFP-LDLR levels.

“levels of GFP-LDLR were not reduced in cells following co-expression of mutant RNF130C304A (Fig. 2D; IB:GFP lane 8 vs. 6)”

RNF130 overexpression resulted in significantly increased total plasma cholesterol levels.

“RNF130 overexpression resulted in significantly increased total (Fig. 1G) and non-HDL (LDL/VLDL) plasma cholesterol levels (Fig. 1H).”

• AssumptionRests on an assumption: Human RNF130 functions equivalently to mouse RNF130 in regulating LDLR and plasma LDL-C.

Overexpression of RNF130 in Pcsk9-/- mice increased HDL-C levels.

“Overexpression of RNF130 in Pcsk9–/– mice (Fig. 4A–B) resulted in increased plasma total cholesterol (Fig. 4C, 4E), LDL/VLDL-C (Fig. 4D–E), and HDL-C levels (Fig. S4A), with no change in plasma TAG levels (Fig. S4B).”

FLAG-RNF130 overexpression in WT mice increased total cholesterol levels.

“FLAG-RNF130 overexpression in WT mice (Fig. S3A–B) increased total and LDL cholesterol levels (Fig. 3B–C)”

• AssumptionRests on an assumption: Human RNF130 functions equivalently to mouse RNF130 in regulating LDLR and plasma LDL-C.

Overexpression of RNF130 significantly decreased levels of LDLR.

“overexpression of RNF130 (Fig. 1C) significantly decreased levels of LDLR (Fig. 1D–E)”

• AssumptionRests on an assumption: Human RNF130 functions equivalently to mouse RNF130 in regulating LDLR and plasma LDL-C.

When heterozygous Rnf130+/- Tm1a mice were crossed, the observed pup ratio was 1 Rnf130+/+: 2 Rnf130+/-: 0.6 Rnf130-/- (n=35 litters, 204 pups total, mean litter size 5.8 pups per litter).

“When heterozygous Rnf130+/– Tm1a mice were crossed with one another, pups were obtained at the ratio of 1 Rnf130+/+: 2 Rnf130+/–: 0.6 Rnf130–/– (n=35 litters, 204 pups total, mean litter size 5.8 pups per litter) which differs from the expected Mendelian ratio of 1:2:1 (Fig. 5A), suggesting that homozygous deletion of Rnf130 is not well tolerated.”

LDL uptake was significantly reduced in cells expressing RNF130.

“LDL uptake was significantly reduced in cells expressing RNF130”

Co-expression of RNF130 greatly increased ubiquitination of LDLR.

“which was accompanied by greatly increased ubiquitination of LDLR (Fig. 2A; Ubiquitination).”

Loss of RNF130 in mice results in increased hepatic levels of LDLR.

“loss of RNF130 in mice results in increased hepatic levels of LDLR”

• Also stated in: § Results: · boosted

  “in vivo disruption of Rnf130 using antisense oligonucleotides, germline deletion, or AAV-CRISPR results in increased hepatic LDLR abundance and availability”

Plasma lipid analysis in Rnf130-/- mice showed a significant reduction in circulating plasma total cholesterol.

“Nevertheless, importantly plasma lipid analysis in Rnf130–/– mice showed a significant reduction in circulating plasma total cholesterol (Fig. 5C) and these changes appeared to be present in the LDL fraction (Fig. 5D).”

Expression of mutant RNF130C304A failed to alter plasma cholesterol levels.

“expression of mutant RNF130C304A (Fig. S3A–B) failed to alter plasma cholesterol levels (Fig. 3B–C)”

• AssumptionRests on an assumption: Human RNF130 functions equivalently to mouse RNF130 in regulating LDLR and plasma LDL-C.

RNF130 C304A failed to lower LDLR protein levels.

“or lower LDLR protein levels (Fig. 2C; Input).”

WT RNF130 decreased the abundance of GFP-tagged LDLR at the cell surface.

“WT RNF130 decreased the abundance of GFP-tagged LDLR at the cell surface (Fig. 2D; IB:GFP lane 7 vs. 6).”

RNF130 overexpression in Pcsk9-/- mice significantly decreased LDLR protein levels.

“Consistent with data from WT mice (Fig. 1E–F), in the absence of any change in Ldlr mRNA (Fig. 4F), RNF130 overexpression resulted in significantly decreased LDLR protein levels (Fig. 4G).”

RNF130 overexpression in Pcsk9-/- mice did not change Ldlr mRNA levels.

“Consistent with data from WT mice (Fig. 1E–F), in the absence of any change in Ldlr mRNA (Fig. 4F), RNF130 overexpression resulted in significantly decreased LDLR protein levels (Fig. 4G).”

RNF130 increases plasma LDL-C levels.

“and increases plasma LDL-C levels”

• Also stated in: § Results: · plain

  “Overexpression of RNF130 decreases hepatic LDLR and increases plasma LDL-C levels.”

Quantification of mean fluorescent intensity demonstrated a significant reduction in total GFP-tagged LDLR levels with co-expression of RNF130.

“quantification of mean fluorescent intensity demonstrated a significant reduction in GFP fluorescence indicating that total levels of GFP-tagged LDLR were also decreased with co-expression of RNF130 (Fig. 2F).”

There was no significant difference in RNF130 mRNA expression between wildtype and mutant RNF130.

“with no significant difference in RNF130 mRNA expression (Fig. S2B)”

The downstream loxP site required for recombination was not present in any of the ES cell clones analyzed.

“Upon sequencing of the ES cells used to generate the Tm1a mice (Fig. S5A), we discovered that the downstream loxP site (Fig. S5A; solid red triangle) required for these recombination events was not present in any of the clones analyzed, thus explaining the lack of Tm1b and Tm1c mice.”

Body weight of adult Rnf130-/- mice at 10 weeks was not significantly different from heterozygous or wildtype mice.

“Surviving Rnf130–/– Tm1a pups were able to nurse, feed, and mature into adults, and the body weight of adult mice at 10 weeks of age was not significantly different from either heterozygous (Rnf130+/–) or wildtype (Rnf130+/+) mice (Fig. S5C).”

Co-expression of RNF130 dramatically decreased the levels of LDLR at the cell surface by immunocytochemical staining.

“Immunocytochemical staining showed that co-expression of RNF130 dramatically decreased the levels of LDLR at the cell surface (Fig. 2E; middle panel).”

Combined mutation of all four residues (K811/816/830R/C839A) prevented the loss of LDLR-GFP protein.

“and the loss of LDLR-GFP protein (Fig. 2B; Input).”

Overexpression of RNF130 had no effect on levels of additional endocytosed receptors EGFR, TfR, and LRP-1 in vitro.

“overexpression of RNF130 had no effect on levels of additional endocytosed receptors EGFR, TfR, and the related lipoprotein receptor LRP-1 (Fig. S2A)”

RNF130 containing a single point mutation in the RING domain (Cys304 → Ala304) failed to ubiquitinate LDLR.

“RNF130 containing a single point mutation in the RING domain (Cys304 → Ala304) failed to ubiquitinate LDLR (Fig. 2C; Ubiquitination)”

Loss of RNF130 in mice results in reduced plasma LDL-C levels.

“and reduced plasma LDL-C levels”

• Also stated in: § Results: · plain

  “and decreased plasma LDL-C levels”

LDLR is redistributed to intracellular vesicles that co-localize with NPC-1, a marker of late endosomes and lysosomes.

“LDLR is redistributed to intracellular vesicles that co-localize with NPC-1, a marker of late endosomes and lysosomes (Fig. S2E)”

Expression of mutant RNF130C304A failed to alter LDLR protein.

“or LDLR protein (Fig. S3A, C).”

• AssumptionRests on an assumption: Human RNF130 functions equivalently to mouse RNF130 in regulating LDLR and plasma LDL-C.

The observed pup ratio differs from the expected Mendelian ratio of 1:2:1.

“When heterozygous Rnf130+/– Tm1a mice were crossed with one another, pups were obtained at the ratio of 1 Rnf130+/+: 2 Rnf130+/–: 0.6 Rnf130–/– (n=35 litters, 204 pups total, mean litter size 5.8 pups per litter) which differs from the expected Mendelian ratio of 1:2:1 (Fig. 5A), suggesting that homozygous deletion of Rnf130 is not well tolerated.”

Overexpression of RNF130 in WT mice increased LDL/VLDL-C levels by 36%.

“Together these data show that overexpression of RNF130 in WT and Pcsk9–/– mice increased LDL/VLDL-C levels by 36% (Fig. 1H–I) and 70% (Fig. 4D–E), respectively, suggesting that the efficacy of RNF130 for increasing LDL/VLDL-C is enhanced when basal hepatic LDLR levels are elevated.”

FLAG-RNF130 overexpression in WT mice was associated with a decrease in LDLR protein.

“and this was associated with a decrease in LDLR protein (Fig. S3A, C).”

• AssumptionRests on an assumption: Human RNF130 functions equivalently to mouse RNF130 in regulating LDLR and plasma LDL-C.

A truncated mRNA is not the reason for the 30% residual Rnf130 expression.

“Expression analysis of predicted Rnf130 transcripts and the knockout cassette indicate that a truncated mRNA is not the reason for the 30% residual Rnf130 expression (Fig. S5D–F).”

RNF130 reduces LDL uptake.

“and LDL uptake”

• Also stated in: § RNF130 is a RING-dependent E3 ligase that ubiquitinates LDLR and redistributes LDLR away from the plasma membrane · plain

  “LDL uptake was significantly reduced in cells expressing RNF130”

Overexpression of RNF130 in Pcsk9-/- mice increased plasma total cholesterol.

“Overexpression of RNF130 in Pcsk9–/– mice (Fig. 4A–B) resulted in increased plasma total cholesterol (Fig. 4C, 4E), LDL/VLDL-C (Fig. 4D–E), and HDL-C levels (Fig. S4A), with no change in plasma TAG levels (Fig. S4B).”

The 30% knockdown in Rnf130+/- mice is significantly lower than the 50% expected of heterozygous animals.

“Furthermore, Rnf130+/– mice only experienced a 30% knockdown of mRNA expression, significantly lower than the 50% that would be expected of heterozygous animals (Fig. 5B).”

Overexpression of RNF130 in Ldlr-/- mice did not change LDL/VLDL-C.

“LDL/VLDL-C (Fig. 3F, Fig. S3E)”

• AssumptionRests on an assumption: Human RNF130 functions equivalently to mouse RNF130 in regulating LDLR and plasma LDL-C.

FPLC analysis demonstrated a significant increase in the LDL-C fraction.

“FPLC analysis of plasma samples from individual mice demonstrated a significant increase in the LDL-C fraction (Fig. 1I).”

• AssumptionRests on an assumption: Human RNF130 functions equivalently to mouse RNF130 in regulating LDLR and plasma LDL-C.

Rnf130+/- mice experienced only a 30% knockdown of Rnf130 mRNA expression.

“Furthermore, Rnf130+/– mice only experienced a 30% knockdown of mRNA expression, significantly lower than the 50% that would be expected of heterozygous animals (Fig. 5B).”

Levels of FLAG-tagged RNF130 were elevated when the RING domain was mutated, compared to wildtype RNF130.

“the levels of FLAG-tagged RNF130 were elevated when the RING domain was mutated, compared to wildtype RNF130 (Fig. 2C; Input)”

RNF130 reduces plasma membrane LDLR localization.

“reduces plasma membrane LDLR localization”

• Also stated in: § Results: · plain

  “resulting in redistribution of the receptor away from the plasma membrane”

RNF130 is an E3 ubiquitin ligase.

“We demonstrate that RNF130 is an E3 ubiquitin ligase that ubiquitinates LDLR”

Total LDLR protein was decreased in cells co-expressing RNF130.

“total LDLR protein was decreased in cells co-expressing RNF130 (Fig. 2A; Input)”

Overexpression of RNF130 in Pcsk9-/- mice increased plasma LDL/VLDL-C.

“Overexpression of RNF130 in Pcsk9–/– mice (Fig. 4A–B) resulted in increased plasma total cholesterol (Fig. 4C, 4E), LDL/VLDL-C (Fig. 4D–E), and HDL-C levels (Fig. S4A), with no change in plasma TAG levels (Fig. S4B).”

Rnf130-/- mice had 30% residual Rnf130 mRNA expression in liver.

“Gene expression analysis of livers from Rnf130+/+, Rnf130+/–, and Rnf130–/– mice demonstrated that Rnf130–/– mice had 30% residual Rnf130 mRNA expression (Fig. 5B), suggesting that surviving hypomorphic mice have escaped entire loss of Rnf130.”

Hepatic Rnf130 mRNA expression was decreased by 70% in mice treated with Rnf130 AAV-CRISPR.

“Hepatic Rnf130 mRNA expression was decreased by 70% in mice treated with Rnf130 AAV-CRISPR (Fig. 6B).”

Changes in LDLR protein were independent of any change in Ldlr mRNA levels.

“Changes in LDLR protein (Fig. 1D–E) were also independent of any change in Ldlr mRNA levels (Fig. 1F)”

• AssumptionRests on an assumption: Human RNF130 functions equivalently to mouse RNF130 in regulating LDLR and plasma LDL-C.

There was a concomitant increase in LDLR protein expression in Rnf130-/- mice compared to their wild-type littermates.

“Consistent with such a decrease in plasma LDL-C, there was a concomitant increase in LDLR protein expression in Rnf130–/– mice compared to their wild-type littermates (Fig. 5E).”

Multiple additional variants in the RNF130 locus were associated with plasma cholesterol in larger data sets.

“We identified multiple additional variants in the RNF130 locus that were associated with plasma cholesterol (Fig. S7A).”

Rnf130 silencing caused significant decreases in LDL cholesterol.

“Rnf130 silencing caused significant decreases in total (Fig. 7C), LDL (Fig. 7D–E), and HDL (Fig. 7E, Fig. S7J) cholesterol, with no significant change in plasma TAG, or liver enzymes ALT and AST (Fig. S7K–M).”

The decrease in plasma cholesterol was mainly attributable to decreases in the LDL fraction.

“Notably, AAV-CRISPR-mediated decreases in Rnf130 mRNA expression were accompanied by significantly decreased plasma cholesterol levels (Fig. 6C) that was mainly attributable to decreases in the LDL fraction (Fig. S6M).”

INDELs were detected in Rnf130 exon 5 in mice treated with Rnf130 AAV-CRISPR.

“As expected, we detected INDELs in Rnf130 exon 5 in mice treated with Rnf130 AAV-CRISPR (Fig. S6F).”

In the HMDP panel of 120 inbred mouse strains, there was a significant correlation between hepatic mRNA expression of Rnf130 and plasma total cholesterol levels.

“In this panel of mice, we identified significant correlation between hepatic mRNA expression of Rnf130 and plasma lipid traits including total (Fig. S7C) and LDL (Fig. S7D) cholesterol levels.”

Silencing Rnf130 in Ldlr-/- mice had no effect on plasma ALT and AST.

“However, silencing Rnf130 in Ldlr–/– mice had no effect on plasma total and LDL-C concentrations (Fig. 7I–J), plasma HDL-C and TAG (Fig. S7N–O), or plasma ALT and AST (Fig. S7P–Q), confirming that the effects on plasma cholesterol observed with silencing Rnf130 require expression of LDLR.”

Rnf130 silencing caused no significant change in liver enzymes ALT and AST.

“Rnf130 silencing caused significant decreases in total (Fig. 7C), LDL (Fig. 7D–E), and HDL (Fig. 7E, Fig. S7J) cholesterol, with no significant change in plasma TAG, or liver enzymes ALT and AST (Fig. S7K–M).”

Silencing Rnf130 in Ldlr-/- mice had no effect on plasma total and LDL-C concentrations.

“However, silencing Rnf130 in Ldlr–/– mice had no effect on plasma total and LDL-C concentrations (Fig. 7I–J), plasma HDL-C and TAG (Fig. S7N–O), or plasma ALT and AST (Fig. S7P–Q), confirming that the effects on plasma cholesterol observed with silencing Rnf130 require expression of LDLR.”

Rnf130 silencing caused no significant change in plasma TAG.

“Rnf130 silencing caused significant decreases in total (Fig. 7C), LDL (Fig. 7D–E), and HDL (Fig. 7E, Fig. S7J) cholesterol, with no significant change in plasma TAG, or liver enzymes ALT and AST (Fig. S7K–M).”

Rnf130 silencing caused significant decreases in HDL cholesterol.

“Rnf130 silencing caused significant decreases in total (Fig. 7C), LDL (Fig. 7D–E), and HDL (Fig. 7E, Fig. S7J) cholesterol, with no significant change in plasma TAG, or liver enzymes ALT and AST (Fig. S7K–M).”

A 70% reduction in Rnf130 mRNA expression in Rnf130-/- mice was sufficient to significantly decrease LDL-C by 45%.

“Whilst a 70% reduction in Rnf130 mRNA expression in Rnf130–/– mice was sufficient to significantly decrease LDL-C by 45%, the same was not true in heterozygous mice.”

The decreases in plasma LDL-C concentrations were accompanied by significant increases in hepatic LDLR protein levels.

“These decreases in plasma LDL-C concentrations were accompanied by significant increases in hepatic LDLR protein levels (Fig. 7F).”

Hepatic RNF130 expression was significantly elevated in individuals with CAD compared to healthy individuals (p = 7.2e-14).

“We also found that hepatic RNF130 expression was significantly elevated in individuals with CAD compared to healthy individuals (p = 7.2e-14).”

Treatment with Rnf130 ASO for four weeks reduced hepatic Rnf130 mRNA levels by more than 90%.

“In contrast to other genetic approaches to disrupt Rnf130 expression (Fig. 5–6), treatment with Rnf130 ASO for four weeks reduced hepatic Rnf130 mRNA levels by more than 90% (Fig. 7B).”

None of the commercially available antibodies tested were specific for endogenous mouse RNF130 in livers of mice treated with control- or Rnf130-AAV-CRISPR.

“As in Fig. S5, we repeated our protein analysis using all commercially available antibodies in the livers of mice treated with either control- or Rnf130-AAV-CRISPR and found that none were specific for endogenous mouse RNF130 (Fig. S6H–L).”

Rnf130 mRNA expression in ASO treated Ldlr-/- mice was decreased by nearly 90%.

“As observed in wildtype mice treated with Rnf130 ASO (Fig. 7B), Rnf130 mRNA expression in ASO treated Ldlr–/– mice was decreased by nearly 90% (Fig. 7H).”

ASO silencing of Rnf130 resulted in a 50% decrease in LDL-C.

“ASO silencing of Rnf130 resulted in a 50% decrease in LDL-C, a change comparable to inhibiting PCSK940,41, further supporting that RNF130 is a significant regulator of LDLR and LDL-C levels.”

There were increased values for discordant sequences in Rnf130 AAV-CRISPR treated mice compared to control treated animals.

“These analyses also show increased values for discordant sequences in these mice when compared to control treated animals (Fig. S6G), suggesting some INDELs are affecting an area larger than the 8bp measurement window.”

ASO silencing of Rnf130, which induced >90% reduction in hepatic Rnf130 mRNA levels, elicited the largest decrease in plasma LDL-C levels.

“Finally, ASO silencing of Rnf130, which induced >90% reduction in hepatic Rnf130 mRNA levels, elicited the largest decrease in plasma LDL-C levels.”

RNF130 was expressed in a liver gene expression co-regulatory network module that is significantly associated with cardiometabolic outcomes.

“RNF130 was expressed in a liver gene expression co-regulatory network module that is significantly associated with cardiometabolic outcomes (Fig. S7B).”

Silencing Rnf130 in Ldlr-/- mice had no effect on plasma HDL-C and TAG.

“However, silencing Rnf130 in Ldlr–/– mice had no effect on plasma total and LDL-C concentrations (Fig. 7I–J), plasma HDL-C and TAG (Fig. S7N–O), or plasma ALT and AST (Fig. S7P–Q), confirming that the effects on plasma cholesterol observed with silencing Rnf130 require expression of LDLR.”

This ubiquitination of LDLR was dependent on both the catalytic RING domain of RNF130, and specific residues in the cytoplasmic tail of LDLR.

“This ubiquitination of LDLR was dependent on both the catalytic RING domain of RNF130, and specific residues in the cytoplasmic tail of LDLR.”

Rnf130+/- mice had no significant change in plasma LDL-C.

“Rnf130+/– mice had only a 30% reduction in Rnf130 expression, significantly less than the expected 50% reduction, but no significant change in plasma LDL-C.”

Rnf130 silencing caused significant decreases in total cholesterol.

“Rnf130 silencing caused significant decreases in total (Fig. 7C), LDL (Fig. 7D–E), and HDL (Fig. 7E, Fig. S7J) cholesterol, with no significant change in plasma TAG, or liver enzymes ALT and AST (Fig. S7K–M).”

Mice with reduced hepatic Rnf130 expression showed increased LDLR protein levels.

“Consistent with previous data in surviving Rnf130–/– mice (Fig. 5), we show increased LDLR protein levels in mice with reduced hepatic Rnf130 expression (Fig. 6D–E).”

In the HMDP panel, there was a significant correlation between hepatic mRNA expression of Rnf130 and plasma LDL cholesterol levels.

“In this panel of mice, we identified significant correlation between hepatic mRNA expression of Rnf130 and plasma lipid traits including total (Fig. S7C) and LDL (Fig. S7D) cholesterol levels.”

AAV-CRISPR-mediated decreases in Rnf130 mRNA expression were accompanied by significantly decreased plasma cholesterol levels.

“Notably, AAV-CRISPR-mediated decreases in Rnf130 mRNA expression were accompanied by significantly decreased plasma cholesterol levels (Fig. 6C) that was mainly attributable to decreases in the LDL fraction (Fig. S6M).”

RNF130 is a novel post-translational regulator of hepatic LDLR and plasma LDL-C levels.

“In the present study we identify RNF130 as a novel post-translational regulator of hepatic LDLR and plasma LDL-C levels.”

• Also stated in: § Conclusions: · plain

  “Our studies identify RNF130 as a novel post-translational regulator of LDL-C levels via modulation of LDLR availability, thus providing important insight into the complex regulation of hepatic LDLR protein levels.”

The efficacy of RNF130 for increasing LDL/VLDL-C is enhanced when basal hepatic LDLR levels are elevated.

“Together these data show that overexpression of RNF130 in WT and Pcsk9–/– mice increased LDL/VLDL-C levels by 36% (Fig. 1H–I) and 70% (Fig. 4D–E), respectively, suggesting that the efficacy of RNF130 for increasing LDL/VLDL-C is enhanced when basal hepatic LDLR levels are elevated.”

PCSK9 normally binds to the LDLR/LDL-C complex as it is being internalized and signals for the LDLR to be degraded in the lysosome, instead of being recycled back to the plasma membrane.

“PCSK9 normally binds to the LDLR/LDL-C complex as it is being internalized and signals for the LDLR to be degraded in the lysosome, instead of being recycled back to the plasma membrane29.”

• cites [29] Sahng WP, Moon YA, Horton JD. Post-transcriptional regulation of low density lipoprotein receptor protein by proprotein convertase subtilisin/kexin type 9a in mouse liver. J Biol Chem. 2004; 279: 50630– 50638. 15385538Not checked yet

RNF130 has been reported to function as an E3 ubiquitin ligase.

“RNF130 has been reported to function as an E3 ubiquitin ligase8.”

• cites [8] Guais A, Siegrist S, Solhonne B, Jouault H, Guellaën G, Bulle F. h-Goliath, paralog of GRAIL, is a new E3 ligase protein, expressed in human leukocytes. Gene. 2006; 112– 120. 16549277 sourceNot checked — no full text of the source

Surviving hypomorphic Rnf130-/- mice have escaped entire loss of Rnf130.

“Gene expression analysis of livers from Rnf130+/+, Rnf130+/–, and Rnf130–/– mice demonstrated that Rnf130–/– mice had 30% residual Rnf130 mRNA expression (Fig. 5B), suggesting that surviving hypomorphic mice have escaped entire loss of Rnf130.”

Overexpression of RNF130 increases plasma LDL-C by a process dependent on both the expression and availability of hepatic LDLR.

“Together with data from Figure 2, these data demonstrate that overexpression of RNF130 increases plasma LDL-C by a process dependent on both the expression and availability of hepatic LDLR.”

WT RNF130 has proportionally greater effects on LDLR abundance at the cell surface than intracellularly.

“Together, these data suggest that WT RNF130 has proportionally greater effects on LDLR abundance at the cell surface.”

RNF130-mediated ubiquitination of LDLR results in the redistribution of LDLR away from the plasma membrane.

“The data of Fig. 2D–F demonstrate that RNF130-mediated ubiquitination of LDLR results in the redistribution of LDLR away from the plasma membrane.”

RNF130 regulates LDL-C through its function as a ubiquitin ligase.

“these experiments together with the data of Fig. 2, demonstrate that RNF130 regulates LDL-C through its function as a ubiquitin ligase”

• AssumptionRests on an assumption: Human RNF130 functions equivalently to mouse RNF130 in regulating LDLR and plasma LDL-C.

E3-ligase activity of RNF130 is required for the regulation of hepatic LDLR and plasma LDL-C.

“E3-Ligase activity of RNF130 is required for the regulation of hepatic LDLR and plasma LDL-C.”

• Also stated in: § Results: · boosted

  “in vitro ubiquitination assays demonstrate RING-dependent regulation of LDLR abundance at the plasma membrane.”

RNF130 regulates LDLR levels via a post-transcriptional mechanism.

“suggesting RNF130 regulates LDLR levels via a post-transcriptional mechanism.”

• AssumptionRests on an assumption: Human RNF130 functions equivalently to mouse RNF130 in regulating LDLR and plasma LDL-C.

LDLR is required for the RNF130-dependent increase in LDL-C levels in vivo.

“demonstrating that LDLR is required for the RNF130-dependent increase in LDL-C levels in vivo.”

• AssumptionRests on an assumption: Human RNF130 functions equivalently to mouse RNF130 in regulating LDLR and plasma LDL-C.

Homozygous deletion of Rnf130 is not well tolerated.

“When heterozygous Rnf130+/– Tm1a mice were crossed with one another, pups were obtained at the ratio of 1 Rnf130+/+: 2 Rnf130+/–: 0.6 Rnf130–/– (n=35 litters, 204 pups total, mean litter size 5.8 pups per litter) which differs from the expected Mendelian ratio of 1:2:1 (Fig. 5A), suggesting that homozygous deletion of Rnf130 is not well tolerated.”

Viral loads were similar in both control and Rnf130 AAV-CRISPR groups.

“These data confirm that viral loads were similar in both groups.”

The increase in hepatic LDLR and decrease in plasma LDL-C after treatment with ASO is comparable to a recently reported gain-of-function mutation in LDLR, which caused a 74% decrease in LDL-C in humans.

“The increase in hepatic LDLR and decrease in plasma LDL-C after treatment with ASO is comparable to a recently reported gain-of-function mutation in LDLR46, which caused a 74% decrease in LDL-C in humans.”

• cites [46] Björnsson E, Gunnarsdóttir K, Halldórsson GH, Sigurðsson Á, Árnadóttir GA, Jónsson H, Ólafsdóttir EF, Niehus S, Kehr B, Sveinbjörnsson G, Lifelong Reduction in LDL Cholesterol Due to a Gain-of-Function Mutation in LDLR. Circ Genomic Precis Med. 2020; CIRCGEN.120.003029. 33315477 sourceNot checked — no full text of the source

RNF130 and IDOL may have important, contrasting roles in the regulation of hepatic LDLR and plasma LDL-C.

“These differences suggest that RNF130 and IDOL may have important, contrasting roles in the regulation of hepatic LDLR and plasma LDL-C.”

RNF130's effects on LDLR protein are independent of PCSK9.

“Our finding that overexpression of RNF130 is able to decrease LDLR levels (and concomitantly increase plasma LDL-C levels) in Pcsk9–/– mice suggests that RNF130’s effects on LDLR protein are independent of PCSK9.”

The effects on plasma cholesterol observed with silencing Rnf130 require expression of LDLR.

“However, silencing Rnf130 in Ldlr–/– mice had no effect on plasma total and LDL-C concentrations (Fig. 7I–J), plasma HDL-C and TAG (Fig. S7N–O), or plasma ALT and AST (Fig. S7P–Q), confirming that the effects on plasma cholesterol observed with silencing Rnf130 require expression of LDLR.”

Some residual expression of Rnf130 is necessary for survival.

“Whole body disruption of Rnf130 expression was incomplete and surviving pups exhibited only a 70% decrease in Rnf130 mRNA, suggesting that some residual expression of Rnf130 is necessary for survival.”

RNF130 is a novel post-translational regulator of hepatic LDLR abundance and activity.

“We have identified RNF130 as a novel post-translational regulator of hepatic LDLR abundance and activity, that may have important implications for targeting the LDLR pathway to lower plasma LDL-C levels.”

Rnf130 regulates cholesterol levels in a dose-dependent manner.

“Taken together, these data suggest that Rnf130 regulates cholesterol levels in a dose-dependent manner.”

The RNF130 locus was previously linked with plasma LDL-C levels in a small population (8,090 individuals) of African Americans.

“The RNF130 locus was previously linked with plasma LDL-C levels in a small population (8,090 individuals) of African Americans33.”

• cites [33] Lettre G, Palmer CD, Young T, Ejebe KG, Allayee H, Benjamin EJ, Bennett F, Bowden DW, Chakravarti A, Dreisbach A, Genome-Wide association study of coronary heart disease and its risk factors in 8,090 african americans: The nhlbi CARe project. PLoS Genet. 2011; PMC3037413 21347282 sourceNot checked yet

  Checked before — its earlier verdict is held back until a new check runs.

Loss of RNF130 is not well tolerated.

“Additionally, currently no homozygotes have been identified for these mutations, suggesting that loss of RNF130 is not well tolerated.”

The RNF130-LDLR pathway could be targeted to enhance LDL clearance from the circulation.

“We further provide pre-clinical evidence that the RNF130-LDLR pathway could be targeted to enhance LDL clearance from the circulation.”