Kidney Week

Abstract: SA-OR15

The Methionine-Mettl3-RNA Methylation Axis Is Essential for Nephrogenesis

Session Information

• Genetics, Development, and Therapies
  November 05, 2022 | Location: W308, Orange County Convention Center‚ West Building
  Abstract Time: 05:06 PM - 05:15 PM

Category: Development‚ Stem Cells‚ and Regenerative Medicine

• 500 Development‚ Stem Cells‚ and Regenerative Medicine

Authors

• Ramalingam, Harini, The University of Texas Southwestern Medical Center Department of Internal Medicine, Dallas, Texas, United States

Harini Ramalingam, PhD

• Alvarez, Jesus A., The University of Texas Southwestern Medical Center Department of Internal Medicine, Dallas, Texas, United States

Jesus A. Alvarez,

• Grilli, Elyse Dyan, The University of Texas Southwestern Medical Center Department of Internal Medicine, Dallas, Texas, United States

Elyse Dyan Grilli,

• Carroll, Thomas J., The University of Texas Southwestern Medical Center Department of Internal Medicine, Dallas, Texas, United States

Thomas J. Carroll,

• Patel, Vishal, The University of Texas Southwestern Medical Center Department of Internal Medicine, Dallas, Texas, United States

Vishal Patel,

Background

RNAs undergo dynamic chemical changes, which can impact their processing, stability, or translational efficiency, a phenomenon termed ‘epitranscriptomics’. N6-methyladenosine (m⁶A) is the most abundant eukaryotic RNA chemical modification. Mettl3, an mRNA methyltransferase, mediates the m⁶A reaction by transferring an activated methyl group from s-adenosyl methionine (SAM) to adenosine. A major contributor of renal failure is low ‘nephron endowment’, which chiefly occurs due to defects in the self-renewal or the differentiation of the nephron progenitors (NPs). Here, we studied whether the methionine-RNA methylation pathway plays a role in the renewal and differentiation of NPs.

Methods

We examined Mettl3 expression in the NP lineage of developing kidneys. We generated mice to conditionally ablate or overexpress Mettl3 in the NP lineage. We used a Mettl3-specific inhibitor or activator to pharmacologically modulate the m⁶A pathway in ex vivo kidney cultures and isolated primary NP cultures. We also tested whether methionine and SAM impact NP fate.

Results

We find that the Mettl3-m⁶A pathway expression is higher in differentiated NPs compared to renewing NPs. Six2/cre-mediated Mettl3 deletion blocks NP differentiation leading to the accumulation of renewing NPs and a markedly lower nephron count. In contrast, deleting Mettl3 subsequent to NP differentiation, using Wnt4/cre, has no impact on kidney development. Conversely, Six2/cre-mediated Mettl3 overexpression results in precocious and ectopic differentiation leading to the premature depletion of renewing NPs. Similarly, acute pharmacological Mettl3 inhibition or methionine depletion blocks NP differentiation whereas acute Mettl3 activation or higher SAM availability has an opposite effect in cultures. Moreover, methionine/SAM promotes Mettl3 expression in NPs, and regulates NP fate through Mettl3. Finally, subthreshold activity of the Mettl3-m⁶A pathway appears to induce both NP renewal and differentiation leading to enhanced nephrogenesis in ex vivo kidney cultures and in kidney organoids.

Conclusion

Our work uncovers RNA methylation as a novel stem cell pathway in metanephros development and links methionine utilization to m⁶A chemical modification in NP fate commitment.

Funding

• NIDDK Support