Maurice Swanson

Recognize hnRNP-M3 As Well

Heterogeneous nuclear ribonucleoproteins (hnRNPs), which directly bind to nascent RNA polymerase II transcripts, play an important role in both transcript-specific packaging and alternative splicing of pre-mRNAs. These "M proteins" are pre-mRNA-binding proteins in vivo, and they bind to poly(G) and poly(U) RNA homopolymers in vitro. hnRNP-M4 is the largest M protein, with 729 amino acids and a molecular mass of about 77 kD. The M4 protein contains 3 ribonucleoprotein consensus sequence RNA-binding domains of approximately 90 amino acids each, a glycine- and methionine-rich region, and an MR (methionine/arginine) repeat motif. It also contains 9 potential casein kinase type II phosphorylation sites and a tyrosine phosphorylation site. Two-dimensional gels showed multiple M4 isoforms, which may be the result of phosphorylation. This antibody also recognizes hnRNP-M3 as well.

Myotonic Dystrophy Type 1 Mouse Model as a Tool for Drug Development and Research

Myotonic dystrophy type 1 (DM1) is an inherited, multi-system disorder, affecting 1 in 8,500 individuals worldwide. However, no effective therapies are currently available for this disease. Clinical symptoms may vary from almost asymptomatic to progressive skeletal muscle wasting, repetitive muscle contractions (myotonia), early onset particulate cataracts, insulin resistance and cardiomyopathy. DM1 pathogenesis is characterized by abnormal expansion of trinucleotide CTG repeat in the gene DMPK on chromosome 19. Even though the mutation responsible for the clinical manifestations of DM1 was identified in 1992, development of effective treatments still requires elucidation of molecular mechanisms.

Researchers at the University of Florida have isolated myoblasts from a Dmpk CTG480 knock-in mouse model for DM1. This cell model serves as an invaluable tool for the study of DM1 pathogenesis and as a translational platform for therapeutic drug development.

Application

A Dmpk CTG480 knock-in mouse platform for therapeutic drug development and for elucidating molecular mechanisms for DM1

Advantages

• A mouse model with a large CTG trinucleotide repeat expansion in the Dmpk 3’ untranslated region, which is the location of this mutation in human DM1 patients
• The spatial and temporal gene expression pattern of this modified Dmpk gene is similar to that observed in DM1 patients
• Serves as a tool to assess the in vivo effects of therapeutic compounds on the expansion mutations that cause DM1

Technology

Dmpk CTG480 knock-in mice were generated using rolling circle amplification, to generate large uninterrupted CTG repeats in vitro, followed by CRISPR/Cas9 genome editing in zygotes. These mice express the CTG480 expansion mutation under the control of the Dmpk promoter and therefore are a mouse model for the neuromuscular disease myotonic dystrophy type 1, the most common form of adult-onset muscular dystrophy.

Recognizes Both Human and Mouse CUGBP2/ETR-3 Proteins but Does Not Bind to Other CELF Family Members

CUGBP2/ETR-3 is a member of the CELF family of pre-mRNA alternative splicing factors which play an important role in the appearance of specific protein isoforms during postnatal development. The CELF family has also been implicated in the neuromuscular disease myotonic dystrophy. The monoclonal antibody was generated against a purified human CUGBP2/ETR-3 protein fragment fused to GST and recognizes both human and mouse CUGBP2/ETR-3 proteins (elav-type RNA-binding protein), but does not bind to other members of the CELF family.

Journal article: http://onlinelibrary.wiley.com/doi/10.1002/dvdy.20382/abstract;jsessionid=C203091EB01A61DC4DBC604B10C9378B.d02t03 (Wiley Online Library subscription required)

Myoblasts Isolated from a Myotonic Dystrophy Type 1 Mouse Model Serve as a Platform for Drug Development and Research

These Dmpk CTG480 knockin mouse myoblasts are isolated from Dmpk CTG480 knockin mice for use as a cell model for myotonic dystrophy type 1. Myotonic dystrophy type 1 (DM1) is an inherited, multi-system disorder, affecting 1 in 8500 individuals worldwide. However, no effective therapies are available for this disease. Clinical symptoms may vary from almost asymptomatic to progressive skeletal muscle wasting, repetitive muscle contractions (myotonia), early onset particulate cataracts, insulin resistance and cardiomyopathy. DM1 pathogenesis is characterized by abnormal expansion of trinucleotide CTG repeat in the gene DMPK on chromosome 19. Even though researchers identified the mutation responsible for the clinical manifestations of DM1 in 1992, development of effective treatments still requires elucidation of molecular mechanisms.

Researchers at the University of Florida have isolated myoblasts from a Dmpk CTG480 knockin mouse model for DM1. This cell model serves as an invaluable tool for the study of DM1 pathogenesis and as a translational platform for therapeutic drug development.

Application

A Dmpk CTG480 knockin myoblast platform for therapeutic drug development and for elucidating molecular mechanisms for DM1

Advantages

• Contains a CTG trinucleotide repeat expansion in Dmpk, which is the gene affected in human DM1 patients
• Serves as a tool to assess the cellular effects of therapeutic compounds on mutations associated with DM1
• Is a cost-effective tool for basic and therapeutic research, serving to help understand molecular mechanisms of DM1 RNA-toxicity and the effects of trinucleotide expansions on cellular pathways

Technology

Primary myoblasts are isolated from a Dmpk CTG480 knockin mouse model of DM1.

PUB1, targeting protein that aids in RNA translation and export

Polyuridylate-binding protein (PUB1) is a member of the ribonuclear protein consensus sequence family of RNA-binding proteins and is structurally related to the human heterologous nuclear RNA-binding proteins (hnRNP) M proteins. PUB1 is localized in a non-uniform pattern throughout both the nucleus and cytoplasm. Antibodies were raised against a preparation of yeast proteins UV crosslinked to polyadenylated RNA. Journal articles: http://mcb.asm.org/content/13/10/6102.long (Molecular and Cell Biology subscription required) http://mcb.asm.org/content/25/13/5499.long (Molecular and Cell Biology subscription required)

PUB 2, targeting protein that aids in RNA translation and export

Polyuridylate-binding protein (PUB2) is also a member of the ribonuclear protein consensus sequence family of RNA-binding proteins and is structurally related to the human heterologous nuclear RNA-binding proteins (hnRNP) M proteins. PUB2 has partially overlapping function with Pub1. Antibodies were raised against a preparation of yeast proteins UV crosslinked to polyadenylated RNA. Journal articles: http://jcb.rupress.org/content/127/5/1173.long (Journal of Cell Biology subscription required) http://www.nature.com/emboj/journal/v21/n7/full/7594399a.html (EMBO Journal subscription required)

Pab1p, targeting protein involved in the construction of mRNA molecules

Pab1p is a protein which binds poly-A tails of yeast mRNA molecules, and is known as polyA binding protein. It is also known as PAB1, ACBP, CST11 and MRNP and the systematic name is YER165w. It is the only yeast poly-A binding protein, and is found in both the cytoplasm and nucleus of yeast cells. The protein has a molecular weight of 64kDa and antibodies were raised against a preparation of yeast proteins UV crosslinked to polyadenylated RNA. Journal article: http://www.nature.com/emboj/journal/v21/n7/full/7594399a.html (EMBO Journal subscription required)

Nab1p, targeting a DNA binding protein in yeast

Mammalian protein arginine methyltransferase (PRMT1) is related to a protein arginine methyltransferase (RMT1) in yeast. This same yeast gene has been independently isolated as an hnRNP (heterogeneous nuclear ribonucleoprotein particle) methyltransferase (HMT1). The substrate specificity of recombinant Rmt1p/Hmt1p indicates that this enzyme is a protein methyltransferase I that methylates glycine/arginine-rich domains. Nab1p is one of the “heterogeneous nuclear RNA” or hnRNA binding proteins of yeast. Nab1p is also sometimes referred to as Npl3p, MTR13p, MTS1p and NOP3p and the systematic name is YDR432W. Nab1p is localized in the nucleus where it has a reticuled expression quite different from, for example Nop1p/fibrillarin. The antibody was raised against a preparation of yeast proteins UV crosslinked to polyadenylated RNA. Journal articles: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC19378/ http://jcb.rupress.org/content/127/5/1173.long (Journal of Cell Biology subscription required)

Nab2p, targeting an RNA binding protein that regulates mRNA within cells

Nab2p is a nuclear polyadenylated RNA-binding protein which autoregulates mRNA levels. It is related to human hnRNPs and has nuclear localization signal sequence that binds to Kap104p; required for poly(A) tail length control and nuclear mRNA export. Journal articles: http://www.jbc.org/content/280/26/24532.long (Journal of Biological Chemistry subscription required) http://www.nature.com/emboj/journal/v21/n7/full/7594399a.html (EMBO Journal subscription required) http://jcb.rupress.org/content/127/5/1173.long (Journal of Cell Biology subscription required)

Nab3p, targeting a DNA binding protein that regulates production of mRNA

Nab3p is a single-stranded DNA binding protein (also known as Hmd1p); acidic ribonucleoprotein; required for termination of non-poly(A) transcripts and efficient splicing; interacts with Nrd1p. The antibody was raised against recombinant Nab3p-maltose binding protein fusion protein. Journal articles: http://jcb.rupress.org/content/127/5/1173.long (Journal of Cell Biology subscription required)

Nab4p, targeting protein that binds RNA for gene expression

Nab4p is a nuclear polyadenylated RNA-binding protein also known as Hrp1p. It is a subunit of cleavage factor I, a five-subunit complex required for the cleavage and polyadenylation of pre-mRNA 3' ends. The antibody was raised against a Nab4p-GST fusion protein. Journal articles: http://www.jbc.org/content/280/26/24532.long (Journal of Biological Chemistry subscription required) http://www.nature.com/emboj/journal/v17/n24/abs/7591431a.html (EMBO Journal subscription required)