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Polypyrimidine Tract Binding Proteins are essential for B cell development

Elisa Monzón-Casanova, Louise S. Matheson, Kristina Tabbada, Kathi Zarnack, Christopher W. J. Smith, Martin Turner

Preprint posted on September 14, 2019 https://www.biorxiv.org/content/10.1101/769141v1

Article now published in eLife at http://dx.doi.org/10.7554/eLife.53557

B cell stutter: B cells fail to develop in PTBP1/2 double knock outs

Selected by Jonny Coates

Categories: cell biology, immunology

Context and background

The adaptive arm of the immune system is comprised of two main cell types: T cells and B cells. Following an immune challenge, B cells rapidly increase production of antibodies, which bind to target antigens located on cells or viruses. Once this binding occurs, these cells are more efficiently cleared by other immune cells through processes such as phagocytosis. Antibodies are proteins comprised of two chains, a heavy chain and a light chain. Within this, antibodies have two regions, designated constant and variable. This structure gives antibodies a classical Y shape with the variable regions located at the tips of the Y where they bind target antigens.

B cells develop in the bone marrow of humans and mice. Early B cell development is characterised by the random rearrangement of the genes that create the antibody variable region, a process called VDJ recombination. The first step of this rearrangement occurs in the pre-pro B cell. The next step occurs in the pro-B cell. At this point, the antibody heavy chains have rearranged but the light chains have not. During the late pre-B cell stages, the light chains undergo a similar rearrangement. Once both chains are formed, the immature B cell undergoes selection to prevent auto-reactivity and only cells that can’t bind self-antigens are released into the periphery (Fig 1.).

Figure 1. B cell development. Reproduced from the preprint (cropped from supplemental figure 1) under a CC BY-NC-ND 4.0 license.

This preprint focusses on polypyrimidine tract binding proteins (PTBP1/2) which bind RNA. PTBP proteins control alternative splicing and mRNA stability (1). In B cells, PTBP1 has been shown to be required for efficient antibody production (2) and to be responsible for the germinal centre B cell response (3). However, there have been limited studies investigating the role of PTBP’s in B cell development. In this preprint, the authors find that PTBP is required for proper B cell development and fidelity of the transcriptome.

Key findings

  1. Loss of PTBP1 and PTBP2 prevents B cell development

Utilising genetic knock-outs of either PTBP1 (sKO) or double knock-outs of PTBP1 and PTBP2 (dKO), the authors assessed the presence of mature B cells using flow cytometry. They demonstrated that, in the dKO, there is a lack of mature B cells but this defect is not observed in the PTBP1 sKO. To address why there were no mature B cells, the authors next assessed B cell development, utilising flow cytometry to visualise different B cell developmental stages. In doing so, the authors find that there is a block in B cell development at the pro-B cell stage in the dKO cells.

  1. B cells lacking PTBP1 and PTBP2 demonstrate cell cycle defects

To explore the underlying cause in the block in B cell development, the authors next investigated cell cycle progression. The authors sequentially labelled cells with EdU and BrdU to label DNA synthesis in order to assess cell cycle stage (Fig 2). The authors found that dKO cells progressed through the S phase better than PTBP1 sKO cells. Further investigation revealed that in the dKO cells, there was a failure to progress to mitosis, with more cells “stuck” in the G2 phase of the cell cycle. This data suggests that the reason for the defect in B cell development is a failure to progress through the cell cycle.

Figure 2. Labelling of cells to assess cell cycle progression. Reproduced from the preprint (cropped from figure 2) under a CC BY-NC-ND 4.0 license.

Cyclin dependent kinases (CDKs) are responsible for controlling the cell cycle. As PTBP proteins modulate mRNA splicing and stability, it is possible that the cell cycle defect is a result of defects in the expression of CDKs. By undertaking an RNAseq experiment, the authors assessed the levels of gene expression of CDK inhibitors. A number of CDK inhibitors were found to be decreased in the dKO cells suggesting that there was a failure in the cell cycle regulation. To investigate this further, the authors assessed the activity of CDKs. This revealed that the dKO cells not only had lower levels of CDK inhibitor expression but also a reduced activity of the CDKs.

Together, this data demonstrates that in PTBP proteins control a host of factors that are essential for avoiding a G2 block and in to promote B cell development (Fig 3.).

Figure 3. Schematic of the main findings of this preprint. Reproduced from the preprint (cropped from figure 2) under a CC BY-NC-ND 4.0 license.

Why I chose this paper

I enjoy maintaining wide interests across immunology. My previous and current work does not expose me to B cells and therefore preLighting an article on B cell biology allows me to delve into a cell type I’m not too familiar with. As the other main component of the adaptive immune response, B cells are hugely important to normal immune system functioning. PTBP proteins are relatively underexplored in B cells and so this is an important paper in uncovering the roles that these protein have in B cell development.

Open questions

  1. The authors demonstrate that pro-B cells express low levels of PTBP2 (supplemental figures). However, they do not investigate levels of PTBP2 in the PTBP1 sKO cells. PTBP1 suppresses PTBP2 so is it possible that there is a compensation/increased expression of PTBP2 when PTBP1 is lost? Additionally, are the authors confident that the low levels of PTBP2 present in B cells are not the true reason for their observations rather than PTBP1? Single KO of PTBP2 would help to answer this question and provide greater strength to the observations made in this preprint.
  1. Is there a role for PTBP3 in B cell development, or any other B cell functions? The authors do not knock out PTBP3 and therefore if this does have a role in B cell development this could explain some of the results.
  1. What is the consequence of the failure of B cells to develop? Is there an associated immunodeficiency? Is the antibody production significantly decreased in these mice when challenged with an appropriate stimulant?
  1. The authors argue that PTBP2 is not expressed in B cells. However, this raises the question of what the reason is for the observed effects on B cells in the double KO cells and why do PTBP1 sKO cells not have the same defects? Is there an interaction between PTBP1 and PTBP2? Again, single KO of PTBP2 would help to answer this question.
  1. Other than modulating CDKs, how do the authors envisage that PTBP1 is functioning in preventing B cell development? For example, is this effecting any other aspect of B cell biology that may be relevant?

References

  1. Romanelli MG, Diani E, Lievens PM-J. New insights into functional roles of the polypyrimidine tract-binding protein. Int J Mol Sci. 2013 Nov 20;14(11):22906–32.
  2. Sasanuma H, Ozawa M, Yoshida N. RNA-binding protein Ptbp1 is essential for BCR-mediated antibody production. Int Immunol. 2019 Mar 5;31(3):157–66.
  3. Monzón-Casanova E, Screen M, Díaz-Muñoz MD, Coulson RMR, Bell SE, Lamers G, et al. The RNA-binding protein PTBP1 is necessary for B cell selection in germinal centers. Nat Immunol. 2018 Mar;19(3):267–78.

 

 

 

Tags: b cell, b cell development, development, ptbp, rna binding

Posted on: 24th October 2019

doi: https://doi.org/10.1242/prelights.14697

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