C-terminal Glycine-rich Domain Research Articles

Mechanism of the natural product moracin-O derived MO-460 and its targeting protein hnRNPA2B1 on HIF-1\u03b1 inhibition

Hypoxia-inducible factor-1α (HIF-1α) mediates tumor cell adaptation to hypoxic conditions and is a potentially important anticancer therapeutic target. We previously developed a method for synthesizing a benzofuran-based natural product, (R)-(-)-moracin-O, and obtained a novel potent analog, MO-460 that suppresses the accumulation of HIF-1α in Hep3B cells. However, the molecular target and underlying mechanism of action of MO-460 remained unclear. In the current study, we identified heterogeneous nuclear ribonucleoprotein A2B1 (hnRNPA2B1) as a molecular target of MO-460. MO-460 inhibits the initiation of HIF-1α translation by binding to the C-terminal glycine-rich domain of hnRNPA2B1 and inhibiting its subsequent binding to the 3’-untranslated region of HIF-1α mRNA. Moreover, MO-460 suppresses HIF-1α protein synthesis under hypoxic conditions and induces the accumulation of stress granules. The data provided here suggest that hnRNPA2B1 serves as a crucial molecular target in hypoxia-induced tumor survival and thus offer an avenue for the development of novel anticancer therapies.

A glycine-rich protein MoGrp1 functions as a novel splicing factor to regulate fungal virulence and growth in Magnaporthe oryzae

Glycine-rich proteins (GRPs) have diverse amino acid sequences and are involved in a variety of biological processes. The role of GRPs in plant pathogenic fungi has not been reported. In this study, we identified and functionally characterized a novel gene named MoGRP1 in Magnaporthe oryzae, which encodes a protein that has an N-terminal RNA recognition motif (RRM) and a C-terminal glycine-rich domain with four Arg-Gly-Gly (RGG) repeats. Deletion of MoGRP1 resulted in dramatic reductions in fungal virulence, mycelial growth, and conidiation. The ΔMogrp1 mutants were also defective in cell wall integrity and in their responses to different stresses. MoGrp1 was localized to the nucleus and was co-immunoprecipitated with several components of the spliceosome, including subunits of the U1 snRNP and U2 snRNP complexes. Moreover, MoGrp1 exhibited binding affinity for poly(U). Importantly, MoGrp1 was responsible for the normal splicing of genes involved in infection-related morphogenesis. Domain deletion assays showed that both the RRM domain and its two adjacent RGG repeats were essential to the full function of MoGrp1. Notably, the nine amino acids between the first and the second RGG repeats were indispensable for nuclear localization and for the biological functions of MoGrp1. Taken together, our data suggest that MoGrp1 functions as a novel splicing factor with poly(U) binding activity to regulate fungal virulence, development, and stress responses in the rice blast fungus.

A Tail of Phage Adhesins

In this issue of Structure, Dunne etal. (2018) unveil the architecture of Salmonella phage S16 adhesin. The structure unravels a beads-on-a-string topology consisting of three domains of which the C-terminal glycine-rich PGII domain, located at the distal tip of the long tail fiber, mediates cell surface attachment and host recognition.

Cytoplasmic Relocalization of TAR DNA-Binding Protein 43 Is Not Sufficient to Reproduce Cellular Pathologies Associated with ALS In vitro.

Mutations in the gene TARDBP, which encodes TAR DNA-binding protein 43 (TDP-43), are a rare cause of familial forms of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). While the majority of mutations are found in the C-terminal glycine-rich domain, an alanine to valine amino acid change at position 90 (A90V) in the bipartite nuclear localization signal (NLS) of TDP-43 has been described. This sequence variant has previously been shown to cause cytoplasmic mislocalization of TDP-43 and decrease protein solubility, leading to the formation of insoluble aggregates. Since the A90V mutation has been described both in patients as well as healthy controls, its pathogenic potential in ALS and FTD remains unclear. Here we compare properties of overexpressed A90V to the highly pathogenic M337V mutation. Though both mutations drive mislocalization of the protein to the cytoplasm to the same extent, M337V produces more significant damage in terms of protein solubility, levels of pathogenic phosphorylation, and formation of C-terminal truncated protein species. Furthermore, the M337V, but not the A90V mutant, leads to a downregulation of histone deacetylase 6 and Ras GTPase-activating protein-binding protein. We conclude that in the absence of another genetic or environmental ‘hit’ the A90V variant is not sufficient to cause the deleterious phenotypes associated with ALS and FTD, despite prominent cytoplasmic protein relocalization of TDP-43.

Structural and biochemical analysis of the Hordeum vulgare L. HvGR-RBP1 protein, a glycine-rich RNA-binding protein involved in the regulation of barley plant development and stress response.

The timing of whole-plant senescence influences important agricultural traits such as yield and grain protein content. Post-transcriptional regulation by plant RNA-binding proteins is essential for proper control of gene expression, development, and stress responses. Here, we report the three-dimensional solution NMR structure and nucleic acid-binding properties of the barley glycine-rich RNA-binding protein HvGR-RBP1, whose transcript has been identified as being >45-fold up-regulated in early—as compared to late—senescing near-isogenic barley germplasm. NMR analysis reveals that HvGR-RBP1 is a multidomain protein comprising a well-folded N-terminal RNA Recognition Motif (RRM) and a structurally disordered C-terminal glycine-rich domain. Chemical shift differences observed in 2D 1H–15N correlation (HSQC) NMR spectra of full-length HvGR-RBP1 and N-HvGR-RBP1 (RRM domain only) suggest that the two domains can interact both in-trans and intramolecularly, similar to what is observed in the tobacco NtGR-RBP1 protein. Further, we show that the RRM domain of HvGR-RBP1 binds single-stranded DNA nucleotide fragments containing the consensus nucleotide sequence 5′-TTCTGX-3′ with low micromolar affinity in vitro. We also demonstrate that the C-terminal glycine-rich (HvGR) domain of Hv-GR-RBP1 can interact nonspecifically with ssRNA in vitro. Structural similarities with other plant glycine-rich RNA-binding proteins suggest that HvGR-RBP1 may be multifunctional. Based on gene expression analysis following cold stress in barley and E. coli growth studies following cold shock treatment, we conclude that HvGR-RBP1 functions in a manner similar to cold-shock proteins and harbors RNA chaperone activity. HvGR-RBP1 is therefore not only involved in the regulation of barley development including senescence, but also functions in plant responses to environmental stress.

The evolutionarily conserved multifunctional glycine-rich RNA-binding proteins play key roles in development and stress adaptation.

The class IV glycine-rich RNA-binding proteins are a distinct subgroup within the heterogenous superfamily of glycine-rich proteins (GRPs). They are distinguished by the presence of an RNA-binding domain in the N-terminus; generally in the form of an RNA-recognition motif (RRM) or a cold-shock domain (CSD). These are followed by a C-terminal glycine-rich domain. Growing evidence suggests that these proteins play key roles in the adaptation of organisms to biotic and abiotic stresses including those resulting from pathogenesis, alterations in the osmotic, saline and oxidative environment and changes in temperature. Similar vertebrate proteins are also cold-induced and involved in, e.g. hibernation, suggesting evolutionarily conserved functions. The class IV RNA-binding GRPs are likely to operate as key molecular components of hormonally regulated development and to work by regulating gene expression at multiple levels by modifying alternative splicing, mRNA export, mRNA translation and mRNA degradation.

Tardbpl splicing rescues motor neuron and axonal development in a mutant tardbp zebrafish

Mutations in the transactive response DNA binding protein-43 (TARDBP/TDP-43) gene, which regulates transcription and splicing, causes a familial form of amyotrophic lateral sclerosis (ALS). Here, we characterize and report the first tardbp mutation in zebrafish, which introduces a premature stop codon (Y220X), eliminating expression of the Tardbp protein. Another TARDBP ortholog, tardbpl, in zebrafish is shown to encode a Tardbp-like protein which is truncated compared with Tardbp itself and lacks part of the C-terminal glycine-rich domain (GRD). Here, we show that tardbp mutation leads to the generation of a novel tardbpl splice form (tardbpl-FL) capable of making a full-length Tardbp protein (Tardbpl-FL), which compensates for the loss of Tardbp. This finding provides a novel in vivo model to study TDP-43-mediated splicing regulation. Additionally, we show that elimination of both zebrafish TARDBP orthologs results in a severe motor phenotype with shortened motor axons, locomotion defects and death at around 10 days post fertilization. The Tardbp/Tardbpl knockout model generated in this study provides an excellent in vivo system to study the role of the functional loss of Tardbp and its involvement in ALS pathogenesis.

TDP-43: A new player on the AD field?

Alzheimer’s disease accounts for an estimated 60–80% of dementia cases, and is the 5th leading cause of death for people aged 65 and older (Heron, 2008). The cognitive decline and amnestic dementia characteristic of the disease result from brain region-specific neuronal dysfunction and death, leading to memory loss, confusion, and changes in behavior, mood and personality. Ultimately, AD patients require help with daily tasks such as eating, bathing and communicating, primarily from unpaid caregivers including family members and friends. In 2009, the contribution of these unpaid caregivers to our nation was valued at $144 billion. As a result of longer life expectancies and the aging baby boomer population, it is expected that by 2050 the number of people aged 65 and older with AD will be between 11–16 million (Hebert, et al., 2003). It is thus imperative that clinical trials toward potential therapeutic targets, which take many years and millions of dollars to reach completion, are initiated now. The regulation of gene expression in the presence and absence of cellular stress is increasingly recognized to be dependent on post-transcriptional mRNA regulation (Liu-Yesucevitz, et al., 2011). In the absence of stress, mature mRNA is transported from the nucleus to the cytosol where it is translated into protein. However, under stress conditions mRNA may be reversibly bound by RNA binding proteins (RBPs) and converted to quiescent cytoplasmic RNA/protein complexes called stress granules (SGs). TAR DNA-binding protein 43 (TDP-43) is a nuclear protein expressed ubiquitously from the Tardbp gene on chromosome 1 (Liu-Yesucevitz, et al., 2010). It contains two highly conserved DNA/RNA recognition motifs, followed by a C-terminal glycine-rich domain and nuclear localization and export signals, which allow TDP-43 to be continuously shuttled between the nucleus and the cytoplasm. Under normal conditions, the predominant function for TDP-43 identified to date is regulation of nuclear transcription, splicing and stability of RNA transcripts (Kumar-Singh, 2011). However, during stress TDP-43 is sequestered in the cytoplasm as a component of insoluble protein aggregates. TDP-43 may also become cleaved by activated caspases, and the phosphorylated C-terminus has become a hallmark of TDP-43 proteinopathy (Liu-Yesucevitz, et al., 2010, Toh, et al., 2011). Although initially recognized for its role in repressing the human immunodeficiency viruse type 1 (HIV-1) gene (Ou, et al., 1995), recent advances in molecular genetics have identified over 30 mutations in TDP-43 that are linked to amyotrophic lateral sclerosis (ALS) and fronto-temporal lobar dementia (FTLD) (Lagier-Tourenne and Cleveland, 2009), suggesting a causative role for the protein in disease development. Indeed, TDP-43 inclusions occur primarily in degenerating motor neurons of the brain and spinal cord. Thus, RBPs have emerged as an important player in neurodegenerative processes, and are a potential targets for therapeutic development.

Requirements for Stress Granule Recruitment of Fused in Sarcoma (FUS) and TAR DNA-binding Protein of 43 kDa (TDP-43)

Cytoplasmic inclusions containing TAR DNA-binding protein of 43 kDa (TDP-43) or Fused in sarcoma (FUS) are a hallmark of amyotrophic lateral sclerosis (ALS) and several subtypes of frontotemporal lobar degeneration (FTLD). FUS-positive inclusions in FTLD and ALS patients are consistently co-labeled with stress granule (SG) marker proteins. Whether TDP-43 inclusions contain SG markers is currently still debated. We determined the requirements for SG recruitment of FUS and TDP-43 and found that cytoplasmic mislocalization is a common prerequisite for SG recruitment of FUS and TDP-43. For FUS, the arginine-glycine-glycine zinc finger domain, which is the protein's main RNA binding domain, is most important for SG recruitment, whereas the glycine-rich domain and RNA recognition motif (RRM) domain have a minor contribution and the glutamine-rich domain is dispensable. For TDP-43, both the RRM1 and the C-terminal glycine-rich domain are required for SG localization. ALS-associated point mutations located in the glycine-rich domain of TDP-43 do not affect SG recruitment. Interestingly, a 25-kDa C-terminal fragment of TDP-43, which is enriched in FTLD/ALS cortical inclusions but not spinal cord inclusions, fails to be recruited into SG. Consistently, inclusions in the cortex of FTLD patients, which are enriched for C-terminal fragments, are not co-labeled with the SG marker poly(A)-binding protein 1 (PABP-1), whereas inclusions in spinal cord, which contain full-length TDP-43, are frequently positive for this marker protein.

Heterogeneous nuclear ribonucleoprotein hrp36 acts as an alternative splicing repressor in Litopenaeus vannamei Dscam

Heterogeneous nuclear ribonucleoproteins (hnRNPs) are highly conserved from nematode to mammal because they play an important role in several aspects of pre-mRNA maturation, including RNA packaging and alternative splicing. In Drosophila, the hnRNP A1 homolog hrp36 regulates alternative splicing in several genes, including the Down syndrome cell adhesion molecule (Dscam), which produces tens of thousands of isoforms from one locus. In this study, the first hrp36 gene was identified and characterized from Litopenaeus vannamei ( Lvhrp36). Its open reading frame (ORF) contains 1101 bp encoding 366 amino acids. The deduced Lvhrp36 protein includes two copies of the RNA recognition motif (RRM), a C-terminal glycine-rich domain (GRD), the highly degenerate RNP consensus sequences RNP-1 and RNP-2, and two RGG boxes. Tissue tropism analysis indicated that Lvhrp36 is expressed ubiquitously and at high levels in most tissues. dsRNA silencing of shrimp Lvhrp36 in vivo induced abnormal exon inclusions in LvDscam, especially in the Ig3 variable region. In the Ig3 region, a total of 14 different combinations were arranged in three different types of abnormal inclusion pattern. This compares to a single combination (one abnormal pattern) in Ig2 and three different combinations (one abnormal pattern) in Ig7. This is the first evidence to suggest that hrp36 may be involved in the regulation of the Ig7 variable region, and it is noteworthy because, at least in Drosophila, there are no hrp36 binding sequences in the Ig7 exon cluster. The above aberrant events were not observed in all of the Lvhrp36-silenced shrimp, and even when they occurred, the normal patterns of inclusion were far more common. We hypothesize that this continued prevalence of normal inclusions was probably due to other unsilenced proteins that were able to rescue Lvhrp36’s functionality. Taken together, our results suggest that Lvhrp36 acts as a splicing repressor that regulates alternative splicing events in the Ig2, Ig3 and Ig7 variable regions of shrimp L. vannamei Dscam.

Mutations in TDP-43 link glycine-rich domain functions to amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is the most common adult motor neuron disease that affects approximately 2/100,000 individuals each year worldwide. Patients with ALS suffer from rapidly progressive degeneration of motor neurons ultimately leading to death. The major pathological features observed in post-mortem tissue from patients with ALS are motor neuron loss, cortical spinal tract degeneration, gliosis and cytoplasmic neuronal inclusions formed by TDP-43 or TAR DNA binding Protein with a molecular mass of 43 kDa, which are now recognized as the signature lesions of sporadic ALS. TDP-43 possesses two RNA binding domains (RBD) and a glycine-rich C terminus classifying it with other heterogeneous nuclear ribonucleoproteins known as 2XRBD-Gly proteins. A number of reports showed that a subset of patients with ALS possess mutations in the TDP-43 (TARDBP) gene. This further strengthens the hypotheses that gain of toxic function or loss of function in TDP-43 causes ALS. Currently, 29 different TARDBP missense mutations have been reported in 51 unrelated sporadic or familial ALS cases and two cases of ALS plus concomitant frontotemporal lobar degeneration with a remarkable concentration of mutations in the C-terminal glycine-rich domain of TDP-43. As these mutations will most certainly be an invaluable tool for the design and implementation of ALS animal and cell models, as well as serve as a platform for exploring the pathobiology of TDP-43, here we summarize the identified pathogenic TARDBP mutations and their potential impact on our understanding of the role of TDP-43 in disease.

Flooding stress-induced glycine-rich RNA-binding protein from Nicotiana tabacum.

A cDNA clone for a transcript preferentially expressed during an early phase of flooding was isolated from Nicotiana tabacum. Nucleotide sequencing of the cDNA clone identified an open reading frame that has high homology to the previously reported glycine-rich RNA-binding proteins. The open reading frame consists of 157 amino acids with an N-terminal RNA-recognition motif and a C-terminal glycine-rich domain, and thus the cDNA clone was designated as Nicotiana tabaccum glycine-rich RNA-binding protein-1 (NtGRP1). Expression of NtGRP1 was upregulated under flooding stress and also increased, but at much lower levels, under conditions of cold, drought, heat, high salt content, and abscisic acid treatment. RNA homopolymer-binding assay showed that NtGRP1 binds to all the RNA homopolymers tested with a higher affinity to poly r(G) and poly r(A) than to poly r(U) and poly r(C). Nucleic acid-binding assays showed that NtGRP1 binds to ssDNA, dsDNA, and mRNA. NtGRP1 suppressed expression of the fire luciferase gene in vitro, and the suppression of luciferase gene expression could be rescued by addition of oligonucleotides. Collectively, the data suggest NtGRP1 as a negative modulator of gene expression by binding to DNA or RNA in bulk that could be advantageous for plants in a stress condition like flooding.

Glorund, a Drosophila hnRNP F/H Homolog, Is an Ovarian Repressor of nanos Translation

Patterning of the anterior-posterior body axis of the Drosophila embryo requires production of Nanos protein selectively in the posterior. Spatially restricted Nanos synthesis is accomplished by translational repression of unlocalized nanos mRNA together with translational activation of posteriorly localized nanos. Repression of unlocalized nanos mRNA is mediated by a bipartite translational control element (TCE) in its 3' untranslated region. TCE stem-loop II functions during embryogenesis, through its interaction with the Smaug repressor. Stem-loop III represses unlocalized nanos mRNA during oogenesis, but trans-acting factors that carry out this function have remained elusive. Here we identify a Drosophila hnRNP, Glorund, that interacts specifically with stem-loop III. We establish that the ability of the TCE to repress translation in vivo reflects its ability to bind Glorund in vitro. These data, together with the analysis of a glorund null mutant, reveal a specific role for an hnRNP in repression of nanos translation during oogenesis.

Gene expression and genetic mapping analyses of a perennial ryegrass glycine-rich RNA-binding protein gene suggest a role in cold adaptation

A perennial ryegrass cDNA clone encoding a putative glycine-rich RNA binding protein (LpGRP1) was isolated from a cDNA library constructed from crown tissues of cold-treated plants. The deduced polypeptide sequence consists of 107 amino acids with a single N-terminal RNA recognition motif (RRM) and a single C-terminal glycine-rich domain. The sequence showed extensive homology to glycine-rich RNA binding proteins previously identified in other plant species. LpGRP1-specific genomic DNA sequence was isolated by an inverse PCR amplification. A single intron which shows conserved locations in plant genes was detected between the sequence motifs encoding RNP-1 and RNP-2 consensus protein domains. A significant increase in the mRNA level of LpGRP1 was detected in root, crown and leaf tissues during the treatment of plants at 4 degrees C, through which freezing tolerance is attained. The increase in the mRNA level was prominent at least 2 h after the commencement of the cold treatment, and persisted for at least 1 week. Changes in mRNA level induced by cold treatment were more obvious than those due to treatments with abscisic acid (ABA) and drought. The LpGRP1 protein was found to localise in the nucleus in onion epidermal cells, suggesting that it may be involved in pre-mRNA processing. The LpGRP1 gene locus was mapped to linkage group 2. Possible roles for the LpGRP1 protein in adaptation to cold environments are discussed.

A cDNA encoding a cold-induced glycine-rich RNA binding protein from Prunus avium expressed in embryonic axes

A cDNA clone encoding a presumed full-length glycine-rich ribonucleic acid (RNA) binding protein was isolated from a λ-ZAP Express cDNA library generated from primarily nondormant Prunus avium (wild cherry) embryonic axes. The cDNA, designated Pa-RRM-GRP1 ( Prunus avium RNA recognition motif glycine-rich protein 1), contains a single N-terminal RNA recognition motif (RRM) and single C-terminal glycine-rich domain. The glycine-rich domain is unusually long at 91 amino acids, 58 of which are glycines. The 534-base pair (bp) open reading frame (ORF) of this clone encodes a 178-amino-acid polypeptide with a predicted molecular weight of 17.33 kDa and p I of 7.84. Comparative sequence alignment of Pa-RRM-GRP1 reveals extensive homology to known and presumed glycine-rich RNA binding proteins from angiosperms and gymnosperms. Genomic Southern blot analysis suggests that this gene exists as a single copy in P. avium. Expression of this gene in P. avium embryonic axes during low-temperature dormancy-breaking treatments was studied and found to be induced by cold (3 °C) using real-time PCR of total cDNA supported by Northern blot analysis of total RNA. Expression dropped during prolonged storage at 3 °C and was reduced to control levels by interruption of cold treatment by warming to 20 °C.

Conservation of structure and cold-regulation of RNA-binding proteins in cyanobacteria: probable convergent evolution with eukaryotic glycine-rich RNA-binding proteins.

The rbp gene family of the cyanobacterium Anabaena variabilis strain M3 consists of eight members that encode small RNA-binding proteins containing a single RNA recognition motif (RRM). Similar genes are found in the genomes of Synechocystis sp. PCC6803, Helicobacter pylori and Treponema pallidum, but are absent from the other completely sequenced prokaryotic genomes. The expression of the rbp genes of Anabaena is induced by low temperature, with the exception of the rbpD gene. We found four stretches of conserved sequences in the 5'-untranslated region of the cyanobacterial rbp genes that are known to be induced by low temperature. The cold-regulated Rbp proteins contain a short C-terminal glycine-rich domain. In this respect, these proteins are similar to plant and mammalian glycine-rich RNA-binding proteins (GRPs), which also contain a single RRM domain with a C-terminal glycine-rich domain and are highly expressed at low temperature. Detailed phylogenetic analysis showed, however, that the cyanobacterial Rbp proteins and the eukaryotic GRPs do not belong to a single lineage, but that the glycine-rich domains are likely to have been added independently. The cold-regulation of both types of proteins is also likely to have evolved independently. Furthermore, the chloroplast RNA-binding proteins are not likely to have originated from the Rbp proteins of endosymbiont cyanobacterium, but are supposed to have diverged from the GRPs. These results suggest that the cyanobacterial Rbp proteins and the eukaryotic GRPs are similar in both structure and regulation, but that this apparent similarity has resulted from convergent evolution.

Crystal structure of human UP1, the domain of hnRNP A1 that contains two RNA-recognition motifs.

Heterogeneous nuclear ribonucleoprotein (hnRNP) A1 is one of the most abundant core proteins of hnRNP complexes in metazoan nuclei. It behaves as a global regulator of alternative pre-mRNA splicing by antagonizing the activities of several serine/arginine-rich splicing factors (SR proteins), resulting in the activation of distal alternative 5' splice sites and skipping of optional exons. Purified hnRNP A1 has nucleic acid annealing activity. The protein also shuttles continuously between the nucleus and the cytoplasm, a process mediated by signals within its C-terminal glycine-rich domain. The N-terminal region of human hnRNP A1, termed unwinding protein 1 (UP1), contains two RNA-recognition motifs (RRMs), RRM1 and RRM2. Understanding the structural elements by which hnRNP A1 interacts with RNA will have broad implications for studies of RNA processing. The crystal structure of UP1 has been determined to 1.9 A resolution. Each RRM independently adopts the characteristic RRM fold, consisting of a four-stranded antiparallel beta-pleated sheet and two alpha helices packed on one side of the beta sheet. The two RRMs are antiparallel and held in close contact, mainly by two Arg-Asp ion pairs. As a result, the two four-stranded beta sheets are brought together to form an extended RNA-binding surface. A segment of the linker connecting the two RRMs is flexible in the absence of bound RNA, but the general location of the linker suggests that it can make direct contacts with RNA. Comparison with other RRM structures indicates that a short 310 helix, found immediately N-terminal to the first beta strand in RRM1, may interact with RNA directly. The RRM is one of the most common and best characterized RNA-binding motifs. In certain cases, one RRM is sufficient for sequence-specific and high affinity RNA binding; but in other cases, synergy between several RRMs within a single protein is required. This study shows how two RRMs are organized in a single polypeptide. The two independently folded RRMs in UP1 are held together in a fixed geometry, enabling the two RRMs to function as a single entity in binding RNA, and so explaining the synergy between the RRMs. The UP1 structure also suggests that residues which lie outside of the RRMs can make potentially important interactions with RNA.

The product of the het-C heterokaryon incompatibility gene of Neurospora crassa has characteristics of a glycine-rich cell wall protein.

Filamentous fungi are capable of hyphal fusion, but heterokaryon formation between different isolates is controlled by specific loci termed het loci. Heterokaryotic cells formed between strains of different het genotype are rapidly destroyed or strongly inhibited in their growth. In Neurospora crassa, at least 11 loci, including the mating type locus, affect the capacity to form a heterokaryon between different isolates. In this report, we describe the molecular characterization of the vegetative incompatibility locus, het-C. The het-COR allele was cloned by genetically identifying the het-C locus in a chromosome walk, and the activity of clones containing the het-COR allele was tested in a functional transformation assay. The het-COR allele encodes a 966-amino acid polypeptide with a putative signal peptide, a coiled-coil motif and a C-terminal glycine-rich domain, similar to glycine-rich domains detected in various extracellular and structural cell envelope proteins. Both the coiled-coil and one-third of the glycine-rich carboxyl terminal domains were required for full het-COR activity. Mutants of het-COR were obtained by repeat-induced point mutation (RIP); these mutants were indistinguishable from wild type during vegetative growth and sexual reproduction but displayed dual compatibility with both of two mutually incompatible het-COR and het-cPA strains.