Human alternative Klotho mRNA is a nonsense-mediated mRNA decay target inefficiently spliced in renal disease

Mencke, Rik; Harms, Geert; Moser, Jill; Meurs, Matijs van; Diepstra, Arjan; Leuvenink, Henri G. D.; Hillebrands, Jan-Luuk

doi:10.1172/jci.insight.94375

Cited by 53 publications

(62 citation statements)

References 68 publications

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“…There is good evidence that αKlotho mRNA may be transcribed but not translated into protein. Mencke et al provided convincing data that the so‐called “spliced Klotho mRNA” is actually destined for degradation by nonsense‐mediated mRNA decay and not translated into protein. RT‐PCR is very sensitive and could amplify non‐specific targets especially when a high cycle number is used.…”

Section: Discussionmentioning

confidence: 99%

Alpha‐Klotho, a critical protein for lung health, is not expressed in normal lung

et al. 2019

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Alpha‐Klotho (αKlotho), produced by the kidney and selected organs, is essential for tissue maintenance and protection. Homozygous αKlotho‐deficiency leads to premature multi‐organ degeneration and death; heterozygous insufficiency leads to apoptosis, oxidative stress, and increased injury susceptibility. There is inconsistent data in the literature regarding whether αKlotho is produced locally in the lung or derived from circulation. We probed murine and human lung by immunohistochemistry (IHC) and immunoblot (IB) using two monoclonal (anti‐αKlotho Kl1 and Kl2 domains) and three other common commercial antibodies. Monoclonal anti‐Kl1 and anti‐Kl2 yielded no labeling in lung on IHC or IB; specific labeling was observed in kidney (positive control) and also murine lungs following tracheal delivery of αKlotho cDNA, demonstrating specificity and ability to detect artificial pulmonary expression. Other commercial antibodies labeled numerous lung structures (IHC) and multiple bands (IB) incompatible with known αKlotho mobility; labeling was not abolished by blocking with purified αKlotho or using lungs from hypomorphic αKlotho‐deficient mice, indicating nonspecificity. Results highlight the need for rigorous validation of reagents. The lung lacks native αKlotho expression and derives full‐length αKlotho from circulation; findings could explain susceptibility to lung injury in extrapulmonary pathology associated with reduced circulating αKlotho levels, for example, renal failure. Conversely, αKlotho may be artificially expressed in the lung, suggesting therapeutic opportunities.

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Section: Discussionmentioning

confidence: 99%

Alpha‐Klotho, a critical protein for lung health, is not expressed in normal lung

et al. 2019

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“…In subsequent work, NMD targets are observed at greater levels in the monosome fraction than in the polysome fractions in yeast (Heyer & Moore, 2016). Additionally, predicted NMD-targeted transcripts were depleted from the polysome fraction, relative to the total cytoplasm or monosome fraction, in human cells (Sterne-Weiler et al, 2013; Floor & Doudna, 2016; Kim et al, 2017; Mencke et al, 2017). These findings are consistent with the evidence that many NMD targets are recognized in the pioneer round of translation (Ishigaki et al, 2001; Chiu et al, 2004).…”

Section: Introductionmentioning

confidence: 99%

Polysome fractionation analysis reveals features important for human nonsense-mediated mRNA decay

Lloyd

French

Brenner

2020

Preprint

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Nonsense-mediated mRNA decay (NMD) is a translation-dependent mRNA surveillance pathway that eliminates transcripts with premature termination codons. Several studies have tried defined the features governing which transcripts are targeted to NMD. However, these approaches often rely on inhibiting core NMD factors, which often have roles in non-NMD processes within the cell. Based on reports that NMD-targeted transcripts are often bound by a single ribosome, we analyzed RNA-Seq data from a polysome fractionation experiment (TrlP-Seq) to characterize the features of NMD-targeted transcripts in human cells. This approach alleviates the need to inhibit the NMD pathway. We found that the exon junction complex (EJC) model, wherein an exon-exon junction located ≥50 nucleotides downstream of a stop codon is predicted to elicit NMD, was a powerful predictor of transcripts with high abundance in the monosome fraction (bound by a single ribosome). This was also true for the presence of an upstream open reading frame. In contrast, as 3’ UTR lengths increase, the proportion of transcripts that are most abundant in the monosome fraction does not increase. This suggests that either longer 3’ UTRs do not consistently act as potent triggers of NMD or that the degradation of these transcripts is mechanistically different to other NMD-targeted transcripts. Of the ribosome-associated transcripts annotated as “non-coding”, we find that a majority are bound by a single ribosome. Many of these transcripts increase in response to NMD inhibition, including the oncogenic SHNG15, suggesting many might be NMD targets. Finally, we found that retained intron transcripts without a premature termination codon are over-represented in the monosome fraction, suggesting an alternative mechanism is responsible for the low level of translation of these transcripts. In summary, our analysis finds that the EJC model is a powerful predictor of NMD-targeted transcripts, while the presence of a long 3’ UTR is not.

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“…8 Apart from the transmembrane form, Klotho has been detected in the urine, blood, and cerebrospinal fluid and may be generated mainly by proteolytic cleavage at the membrane by ADAM metallopeptidase domain proteases, ADAM10 and ADAM17 (soluble form), or through a splicing variant (secreted form), [9][10][11][12][13][14][15] the fate of which has been recently debated since this mRNA may be rapidly degraded. 16 Renal Klotho levels decline in experimental models of Chronic Kidney Disease (CKD). This likely contributes directly to hyperphosphatemia, increased vascular calcification, and cardiac hypertrophy, but potentially also has indirect effects via the parallel elevation of FGF-23.…”

Section: Introductionmentioning

confidence: 99%

Klotho regulation by albuminuria is dependent on ATF3 and endoplasmic reticulum stress

et al. 2019

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Proteinuria is associated with renal function decline and cardiovascular mortality. This association may be attributed in part to alterations of Klotho expression induced by albuminuria, yet the underlying mechanisms are unclear. The presence of albumin decreased Klotho expression in the POD‐ATTAC mouse model of proteinuric kidney disease as well as in kidney epithelial cell lines. This downregulation was related to both decreased Klotho transcription and diminished protein half‐life, whereas cleavage by ADAM proteases was not modified. The regulation was albumin specific since it was neither observed in the analbuminemic Col4α3−/− Alport mice nor induced by exposure of kidney epithelial cells to purified immunoglobulins. Albumin induced features of ER stress in renal tubular cells with ATF3/ATF4 activation. ATF3 and ATF4 induction downregulated Klotho through altered transcription mediated by their binding on the Klotho promoter. Inhibiting ER stress with 4‐PBA decreased the effect of albumin on Klotho protein levels without altering mRNA levels, thus mainly abrogating the increased protein degradation. Taken together, albuminuria decreases Klotho expression through increased protein degradation and decreased transcription mediated by ER stress induction. This implies that modulating ER stress may improve proteinuria‐induced alterations of Klotho expression, and hence renal and extrarenal complications associated with Klotho loss.

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Human alternative Klotho mRNA is a nonsense-mediated mRNA decay target inefficiently spliced in renal disease

Cited by 53 publications

References 68 publications

Alpha‐Klotho, a critical protein for lung health, is not expressed in normal lung

Alpha‐Klotho, a critical protein for lung health, is not expressed in normal lung

Polysome fractionation analysis reveals features important for human nonsense-mediated mRNA decay

Klotho regulation by albuminuria is dependent on ATF3 and endoplasmic reticulum stress

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