Katrina Rothblum

Glypican-1 and α4(V) Collagen Are Required for Schwann Cell Myelination

Schwann cell myelination requires interactions with the extracellular matrix (ECM) mediated by cell surface receptors. Previously, we identified a type V collagen family member, α4(V) collagen, which is expressed by Schwann cells during peripheral nerve differentiation. This collagen binds with high affinity to heparan sulfate through a unique binding motif in the noncollagenous N-terminal domain (NTD). The principal α4(V) collagen-binding protein on the Schwann cell surface is the heparan sulfate proteoglycan glypican-1. We investigated the role of α4(V) collagen and glypican-1 in Schwann cell terminal differentiation in cultures of Schwann cells and dorsal root ganglion neurons. Small interfering RNA-mediated suppression of glypican-1 expression decreased binding of α4(V)-NTD to Schwann cells, adhesion and spreading of Schwann cells on α4(V)-NTD, and incorporation of α4(V) collagen into Schwann cell ECM. In cocultures, α4(V) collagen coassembles with laminin on the surface of polarized Schwann cells to form tube-like ECM structures that are sites of myelination. Suppression of glypican-1 or α4(V) collagen expression significantly inhibited myelination. These results demonstrate an important role for these proteins in peripheral nerve terminal differentiation.

Constitutive Release of α4 Type V Collagen N-terminal Domain by Schwann Cells and Binding to Cell Surface and Extracellular Matrix Heparan Sulfate Proteoglycans

During peripheral nerve development, Schwann cells synthesize collagen type V molecules that contain α4(V) chains. This collagen subunit possesses an N-terminal domain (NTD) that contains a unique high affinity heparin binding site. The α4(V)-NTD is adhesive for Schwann cells and sensory neurons and is an excellent substrate for Schwann cell and axonal migration. Here we show that the α4(V)-NTD is released constitutively by Schwann cells both in culture and in vivo. In cultures of neonatal rat Schwann cells, α4(V)-NTD release is increased significantly by ascorbate treatment, which facilitates collagen post-translational modification and collagen trimer assembly. In peripheral nerve tissue, the α4(V)-NTD is localized to the region of the outer Schwann cell membrane and associated extracellular matrix. The released α4(V)-NTD binds to the cell surface and extracellular matrix heparan sulfate proteoglycans of Schwann cells. Pull-down assays and immunofluorescent staining showed that the major α4(V)-NTD-binding proteins are glypican-1 and perlecan. α4(V)-NTD binding occurs via a mechanism that requires the high affinity heparin binding site and that is blocked by soluble heparin, demonstrating that binding to proteoglycans is mediated by their heparan sulfate chains.

DNA Binding by the Ribosomal DNA Transcription Factor Rrn3 Is Essential for Ribosomal DNA Transcription

Background: Transcription initiation by RNA polymerase I requires protein-protein interactions between Rrn3, polymerase, and core factors. Results: Mutagenesis of a putative DNA binding domain in Rrn3 had no effect on essential protein-protein interactions, but abrogated DNA binding and inactivated Rrn3 function in transcription. Conclusion: DNA binding is essential for Rrn3 to function in transcription. Significance: DNA binding by Rrn3 may provide an additional target to regulate rDNA transcription. The human homologue of yeast Rrn3 is an RNA polymerase I-associated transcription factor that is essential for ribosomal DNA (rDNA) transcription. The generally accepted model is that Rrn3 functions as a bridge between RNA polymerase I and the transcription factors bound to the committed template. In this model Rrn3 would mediate an interaction between the mammalian Rrn3-polymerase I complex and SL1, the rDNA transcription factor that binds to the core promoter element of the rDNA. In the course of studying the role of Rrn3 in recruitment, we found that Rrn3 was in fact a DNA-binding protein. Analysis of the sequence of Rrn3 identified a domain with sequence similarity to the DNA binding domain of heat shock transcription factor 2. Randomization, or deletion, of the amino acids in this region in Rrn3, amino acids 382–400, abrogated its ability to bind DNA, indicating that this domain was an important contributor to DNA binding by Rrn3. Control experiments demonstrated that these mutant Rrn3 constructs were capable of interacting with both rpa43 and SL1, two other activities demonstrated to be essential for Rrn3 function. However, neither of these Rrn3 mutants was capable of functioning in transcription in vitro. Moreover, although wild-type human Rrn3 complemented a yeast rrn3-ts mutant, the DNA-binding site mutant did not. These results demonstrate that DNA binding by Rrn3 is essential for transcription by RNA polymerase I.

α7β1 integrin is a receptor for laminin-2 on Schwann cells

The Schwann cell basal lamina acts as an organizer of peripheral nerve tissue and influences many aspects of cell behavior during development and regeneration. A principal component of the Schwann cell basal lamina is laminin‐2. This study was undertaken to identify Schwann cell receptors for laminin‐2. We found that among several Schwann cell integrins that can potentially interact with laminin‐2, only α7β1 bound to laminin‐2‐Sepharose. Dystroglycan, a non‐integrin Schwann cell receptor for laminin‐2 identified previously, was also found to bind to laminin‐2‐Sepharose. Antibody to the α7 integrin subunit partially inhibited Schwann cell adhesion to laminin‐2. Small interfering RNA‐mediated suppression of either α7 integrin or dystroglycan expression decreased adhesion and spreading of Schwann cells on laminin‐2, whereas knocking down both proteins together inhibited adhesion and spreading on laminin‐2 almost completely. α7 integrin and dystroglycan both colocalized with laminin‐2 containing basal lamina tubes in differentiating neuron–Schwann cell cocultures. The α7β1 integrin also coprecipitates with focal adhesion kinase in differentiating cocultures. These findings strongly suggest that α7β1 integrin is a Schwann cell receptor for laminin‐2 that provides transmembrane linkage between the Schwann cell basal lamina and cytoskeleton.

Selective Inhibition of rDNA Transcription by a Small- Molecule Peptide That Targets the Interface between RNA Polymerase I and Rrn3

The interface between the polymerase I–associated factor Rrn3 and the 43-kDa subunit of RNA polymerase I is essential to the recruitment of Pol I to the preinitiation complex on the rDNA promoter. In silico analysis identified an evolutionarily conserved 22 amino acid peptide within rpa43 that is both necessary and sufficient to mediate the interaction between rpa43 and Rrn3. This peptide inhibited rDNA transcription in vitro, while a control peptide did not. To determine the effect of the peptide in cultured cells, the peptide was coupled to the HIV TAT peptide to facilitate transduction into cells. The wild-type peptide, but not control peptides, inhibited Pol I transcription and cell division. In addition, the peptide induced cell death, consistent with other observations that “nucleolar stress” results in the death of tumor cells. The 22mer is a small-molecule inhibitor of rDNA transcription that is specific for the interaction between Rrn3 and rpa43, as such it represents an original way to interfere with cell growth. Implications: These results demonstrate a potentially novel pharmaceutical target for the therapeutic treatment of cancer cells. Mol Cancer Res; 12(11); 1586–96. ©2014 AACR.

Characterization of the interactions of mammalian RNA polymerase I associated proteins PAF53 and PAF49.

Masami Muramatsus laboratory demonstrated the critical role of RNA polymerase I (Pol I)-associated factor PAF53 in mammalian rRNA transcription. They have also identified a second polymerase associated factor, PAF49. Both PAF49 and PAF53 copurify with that fraction of the RNA polymerase I molecules that can function in transcription initiation in vitro. PAF49 and PAF53 are the mammalian homologues of two subunits of yeast RNA polymerase I, A34.5 and A49, that form a TFIIF-related subcomplex in yeast RNA polymerase I. In light of those publications, we investigated the interactions between various deletion and substitution mutants of mammalian PAF49 and PAF53 with the purpose of identifying those domains of the mammalian proteins that interact. Comparison of our results with structural studies on yeast A34.5 and A49 demonstrates that the yeast and mammalian proteins may in fact share structural similarities. In fact, the deletion mutagenesis data confirmed and extended the structural studies. For example, amino acids 41-86 of PAF49 were sufficient to provide the basis for heterodimerization. In silico structural analysis predicted that this region could assume a structure similar to the homologous region of yeast A34.5. Those similarities are insufficient, by themselves, for the proteins to form interspecific heterodimers. However, substitution of amino acids 52-98 of yeast A34.5 with amino acids 41-86 of mammalian PAF49 resulted in a protein that could heterodimerize with mouse PAF53.