poly(C)-binding Protein Research Articles

Exploration of Potential Broad-Spectrum Antiviral Targets in the Enterovirus Replication Element: Identification of Six Distinct 5' Cloverleaves.

Enterovirus genomic replication initiates at a predicted RNA cloverleaf (5'CL) at the 5' end of the RNA genome. The 5'CL contains one stem (SA) and three stem-loops (SLB, SLC, SLD). Here, we present an analysis of 5'CL conservation and divergence for 209 human health-related serotypes from the enterovirus genus, including enterovirus and rhinovirus species. Phylogenetic analysis indicates six distinct 5'CL serotypes that only partially correlate with the species definition. Additional findings include that 5'CL sequence conservation is higher between the EV species than between the RV species, the 5'CL of EVA and EVB are nearly identical, and RVC has the lowest 5'CL conservation. Regions of high conservation throughout all species include SA and the loop and nearby bases of SLB, which is consistent with known protein interactions at these sites. In addition to the known protein binding site for the Poly-C binding protein in the loop of SLB, other conserved consecutive cytosines in the stems of SLB and SLC provide additional potential interaction sites that have not yet been explored. Other sites of conservation, including the predicted bulge of SLD and other conserved stem, loop, and junction regions, are more difficult to explain and suggest additional interactions or structural requirements that are not yet fully understood. This more intricate understanding of sequence and structure conservation and variability in the 5'CL may assist in the development of broad-spectrum antivirals against a wide range of enteroviruses, while better defining the range of virus isotypes expected to be affected by a particular antiviral.

Iron-tracking strategies: Chaperones capture iron in the cytosolic labile iron pool.

Cells express hundreds of iron-dependent enzymes that rely on the iron cofactors heme, iron-sulfur clusters, and mono-or di-nuclear iron centers for activity. Cells require systems for both the assembly and the distribution of iron cofactors to their cognate enzymes. Proteins involved in the binding and trafficking of iron ions in the cytosol, called cytosolic iron chaperones, have been identified and characterized in mammalian cells. The first identified iron chaperone, poly C-binding protein 1 (PCBP1), has also been studied in mice using genetic models of conditional deletion in tissues specialized for iron handling. Studies of iron trafficking in mouse tissues have necessitated the development of new approaches, which have revealed new roles for PCBP1 in the management of cytosolic iron. These approaches can be applied to investigate use of other nutrient metals in mammals.

Dynamics and energetics of PCBP1 binding to severely oxidized RNA.

Oxidatively generated lesions such as 8-oxo-7, 8-dihydroguanine (8-oxoG) on RNA strands constitute a hallmark marker of the oxidative stress in the cell. Poly-C binding protein 1 (PCBP1) is able to specifically recognize severely damaged RNA strands containing two 8-oxoG lesions separated by five nucleobases, which trigger a signaling pathway leading to cell apoptosis. We apply an in silico protocol based on microsecond timescale all-atom classical molecular dynamics simulations associated with conformational and energy analyses to unveil the specific recognition mechanism at a molecular level. By comparing the RNA and protein behavior for sequences with six different damage profiles, our results highlight an allosteric mechanism, allowing a stronger binding of the oxidized guanine at position 9 only if another 8-oxoG lesion is present at position 15, in full agreement with experiments. We assess the role of lysine K23 and the additional ketone group of the oxidized guanine, thanks to computational site-directed mutagenesis.

Mechanistic insights into poly(C)-binding protein hnRNP K resolving i-motif DNA secondary structures

I-motifs are four-strand noncanonical secondary structures formed by cytosine (C)-rich sequences in living cells. The structural dynamics of i-motifs play essential roles in many cellular processes, such as telomerase inhibition, DNA replication, and transcriptional regulation. In cells, the structural dynamics of the i-motif can be modulated by the interaction of poly(C)-binding proteins (PCBPs), and the interaction is closely related to human health, through modulating the transcription of oncogenes and telomere stability. Therefore, the mechanisms of how PCBPs interact with i-motif structures are fundamentally important. However, the underlying mechanisms remain elusive. I-motif structures in the promoter of the c-MYC oncogene can be unfolded by heterogeneous nuclear ribonucleoprotein K (hnRNP K), a PCBP, to activate its transcription. Here, we selected this system as an example to comprehensively study the unfolding mechanisms. We found that the promoter sequence containing 5 C-runs preferred folding into type-1245 to type-1234 i-motif structures based on their folding stability, which was further confirmed by single-molecule FRET. In addition, we first revealed that the c-MYC i-motif structure was discretely resolved by hnRNP K through two intermediate states, which were assigned to the opposite hairpin and neighboring hairpin, as further confirmed by site mutations. Furthermore, we found all three KH (hnRNP K homology) domains of hnRNP K could unfold the c-MYC i-motif structure, and KH2 and KH3 were more active than KH1. In conclusion, this study may deepen our understanding of the interactions between i-motifs and PCBPs and may be helpful for drug development.

Circular RNA circEsyt2 regulates vascular smooth muscle cell remodeling via splicing regulation.

Circular RNAs (circRNAs) have been recently recognized as playing a role in the pathogenesis of vascular remodeling-related diseases by modulating the functions of miRNAs. However, the interplay between circRNAs and proteins during vascular remodeling remains poorly understood. Here, we investigated a previously identified circRNA, circEsyt2, whose expression is known to be upregulated during vascular remodeling. Loss- and gain-of‑function mutation analyses in vascular smooth muscle cells (VSMCs) revealed that circEsyt2 enhanced cell proliferation and migration and inhibited apoptosis and differentiation. Furthermore, the silencing of circEsyt2 in vivo reduced neointima formation, while circEsyt2 overexpression enhanced neointimal hyperplasia in the injured carotid artery, confirming its role in vascular remodeling. Using unbiased protein-RNA screening and molecular validation, circEsyt2 was found to directly interact with polyC-binding protein 1 (PCBP1), an RNA splicing factor, and regulate PCBP1 intracellular localization. Additionally, circEsyt2 silencing substantially enhanced p53β splicing via the PCBP1-U2AF65 interaction, leading to the altered expression of p53 target genes (cyclin D1, p21, PUMA, and NOXA) and the decreased proliferation of VSMCs. Thus, we identified a potentially novel circRNA that regulated vascular remodeling, via altered RNA splicing, in atherosclerotic mouse models.

MicroRNA-122 and Poly-C Binding Protein 2 Regulate Hepatitis C Replication by Competing for Overlapping Binding Sites

Hepatitis C virus (HCV) is a human pathogen that causes a persistent infection in the liver that requires precise control over viral replication to be maintained. Successful HCV replication is promoted by many host factors; of importance to this study is microRNA-122 (miR-122) and Poly-C Binding Protein-2 (PCBP2). The HCV 5’ UTR contains two miR-122 binding sites near the first stem-loop (SLI) and a PCBP2 binding region that overlaps the second miR-122 binding site. PCBP2 also binds to the 3’ UTR and has been shown to circularize the HCV genome.

Derivation and Characterization of Pcbp1-Deficient Mouse Embryonic Stem Cells

KH-domain PolyC-binding proteins perform many functions ranging from transcription regulation to alternative splicing. There are several evidences of their important role in embryogenesis and in embryonic stem cells (ESCs). ESCs are cultured counterparts of epiblast cells before implantation, and both cell types are in naive state of pluripotency. We have previously shown that members of the KH-domain family—hnRNP-K, Pcbp1 and Pcbp2 proteins—are able to bind to the PolyC-sites within regulatory elements of the Pou5f1 gene that encodes the key pluripotency gatekeeper Oct4. In addition, early lethal phenotype of Pcbp1–/– mouse embryos has been described in literature. In this study, we set to assess role of Pcbp1 in ESCs. To this end, we have obtained null-Pcbp1 ESCs. Contrary to previously observed by us lethal phenotype of hnRNP-K in ESCs, Pcbp1–/– ESCs were viable, continued to express Oct4 and retained overall pluripotent properties. Therefore, the obtained data reveal no critical role of Pcbp1 in self-renewal of naive pluripotent stem cells. Further studies should address whether this is the case during the transition of these cells to the primed state of pluripotency, as well as during the exit from this state due course of differentiation into the three germ layers.

PCBP1 and PCBP2 both bind heavily oxidized RNA but cause opposing outcomes, suppressing or increasing apoptosis under oxidative conditions

PCBP1, a member of the poly(C)-binding protein (PCBP) family, has the capability of binding heavily oxidized RNA and therefore participates in the cellular response to oxidative conditions, helping to induce apoptosis. There are four other members of this family, PCBP2, PCBP3, PCBP4, and hnRNPK, but it is not known whether they play similar roles. To learn more, we first tested their affinity for an RNA strand carrying two 8-oxoguanine (8-oxoG) residues at sites located in close proximity to each other, representative of a heavily oxidized strand or RNA with one 8-oxoG or none. Among them, only PCBP2 exhibited highly selective binding to RNA carrying two 8-oxoG residues similar to that observed with PCBP1. In contrast, PCBP3, PCBP4, and hnRNPK bound RNA with or without 8-oxoG modifications and exhibited slightly increased binding to the former. Mutations in conserved RNA-binding domains of PCBP2 disrupted the specific interaction with heavily oxidized RNA. We next tested PCBP2 activity in cells. Compared with WT HeLa S3 cells, PCBP2-KO cells established by gene editing exhibited increased apoptosis with increased caspase-3 activity and PARP1 cleavage under oxidative conditions, which were suppressed by the expression of WT PCBP2 but not one of the mutants lacking binding activity. In contrast, PCBP1-KO cells exhibited reduced apoptosis with much less caspase-3 activity and PARP cleavage than WT cells. Our results indicate that PCBP2 as well as PCBP1 bind heavily oxidized RNA; however, the former may counteract PCBP1 to suppress apoptosis under oxidative conditions.

Expression patterns of pcbp gene family members during zebrafish embryogenesis

The poly(C)-binding protein (PCBP) family members belong to a subtype of RNA-binding proteins that are ubiquitously expressed with diverse functions. In mammals, PCBP family, also known as hnRNP E family, is composed of four proteins, namely PCBP1, PCBP2, PCBP3 and PCBP4. So far, no study has been documented on the physiological roles of each member in vertebrate development. Here we analysed the spatiotemporal expression patterns of zebrafish (Danio rerio) pcbp2 (identical to pcbp1 and pcbp2 in mammals), pcbp3 and pcbp4 at various stages of zebrafish embryonic development by whole-mount in situ hybridization. Our results revealed that all pcbp genes are maternally expressed, especially pcbp2, which is strongly expressed from the embryogenetic stage to larva. The expression patterns of PCBP members are similar to each other at the very early developmental stage sharing with common strong expression in the intestine, otic vesicle, retina and brain of zebrafish. Subsequently, the messenger RNAs of PCBP members are gradually constrained and highly expressed in intestines of the larvae. Collectively, our study figured out the expression pattern of each PCBP member in diverse organogenesis during embryo development, indicating that PCBP members may play predominant roles in the development of neural and digestive systems to maintain their normal physiological functions. Moreover, the similar expression patterns at the developmental stages and organ types among this family suggest that the aberrant expression of these genes would lead to the neural or intestinal diseases.

Expression Pattern of Major Poly C Binding Protein (Pcbp) Isoforms in Cancer Cell Lines of Cervix, Melanoma and Muscle

Poly (C) binding proteins (PCBPs) are members of sequence specific RNA binding protein family with conserved KH domain. There are four identified isoforms such as Pcbp1 or α-CP1 (α-Complex proteins), Pcbp2 or α-CP2, Pcbp3 or α-CP3 and Pcbp4 or α-CP4. Among them Pcbp1 and Pcbp2 are the most studied and found to be associated with various cellular functions such as transcriptional regulations, translational regulations and mRNA stability. Although two proteins share extensive similarity, they differ in function and localization. Pcbp1 has role in tumorigenesis, and metastasis, which are key phenomena of cancer. Role of pcbp2 has been well documented in the biology of RNA virus, namely translation and replication. Here, we studied expression pattern of Pcbp1 and Pcbp2 in three different cancer cell lines namely HeLa, RD, and A375 originated from different tissues. The results indicate not only differential abundance of these two proteins in three cell lines, but also discordant expression of pcbp1 in mRNA and protein level in three cell lines. The study therefore suggests post-transcriptional regulation of pcbp1 expression in these cell lines.

Metabolic stress activates an ERK/hnRNPK/DDX3X pathway in pancreatic β cells

ObjectivePancreatic β cell failure plays a central role in the development of type 2 diabetes (T2D). While the transcription factors shaping the β cell gene expression program have received much attention, the post-transcriptional controls that are activated in β cells during stress are largely unknown. We recently identified JUND as a pro-oxidant transcription factor that is post-transcriptionally upregulated in β cells during metabolic stress. Here we seek to uncover the mechanisms underlying this maladaptive response to metabolic stress. MethodsRNA-protein and protein-protein interactions were measured using RNA immunoprecipitation and co-immunoprecipitation, respectively, in Min6 cells and mouse islets. Phos-tag analyses were used to assess hnRNPK phosphorylation in primary mouse and human islets and Min6 cells. Translating ribosome affinity purification (TRAP) followed by RT-qPCR was used to identify changes in the ribosome occupancy of mRNAs in Min6 cells. Gene depletion studies used lentiviral delivery of CRISPR-Cas9 to Min6 cells. Apoptosis was measured in primary islets using a cell-permeable dye with a fluorescence readout of activated cleaved caspase-3 and-7. ResultsA de novo motif analysis was performed on a subset of genes previously found to be regulated at the level of ribosome binding during PDX1-deficiency, which identified a poly-cytosine (polyC) motif in the 3′UTR of the transcript encoding JUND. The polyC-binding protein hnRNPK bound to the mRNA encoding JUND, leading us to hypothesize that hnRNPK regulates JUND expression during glucolipotoxicity. Indeed, loss of hnRNPK blocked the post-transcriptional upregulation of JUND during metabolic stress. hnRNPK was phosphorylated in mouse and human islets during glucolipotoxicity and in islets of diabetic db/db mice. The MEK/ERK signaling pathway was both necessary and sufficient for the phosphorylation of hnRNPK, upregulation of JUND levels, and induction of pro-oxidant and pro-inflammatory genes. Further, we identified the RNA helicase DDX3X as a new binding partner for hnRNPK that is required for efficient translation of JUND mRNA. Loss of hnRNPK reduced DDX3X binding to translation machinery, suggesting that these factors cooperate to regulate translation in β cells. ConclusionsOur results identify a novel ERK/hnRNPK/DDX3X pathway that influences β cell survival and is activated under conditions associated with T2D.

A cytosine-rich splice regulatory determinant enforces functional processing of the human α-globin gene transcript

AbstractThe establishment of efficient and stable splicing patterns in terminally differentiated cells is critical to maintenance of specific functions throughout the lifespan of an organism. The human α-globin (hα-globin) gene contains 3 exons separated by 2 short introns. Naturally occurring α-thalassemia mutations that trigger aberrant splicing have revealed the presence of cryptic splice sites within the hα-globin gene transcript. How cognate (functional) splice sites are selectively used in lieu of these cryptic sites has remained unexplored. Here we demonstrate that the preferential selection of a cognate splice donor essential to functional splicing of the hα-globin transcript is dependent on the actions of an intronic cytosine (C)-rich splice regulatory determinant and its interacting polyC-binding proteins. Inactivation of this determinant by mutation of the C-rich element or by depletion of polyC-binding proteins triggers a dramatic shift in splice donor activity to an upstream, out-of-frame, cryptic donor. The essential role of the C-rich element in hα-globin gene expression is supported by its coevolution with the cryptic donor site in primate species. These data lead us to conclude that an intronic C-rich determinant enforces functional splicing of the hα-globin transcript, thus acting as an obligate determinant of hα-globin gene expression.

PolyC-binding proteins enhance expression of the CDK2 cell cycle regulatory protein via alternative splicing.

The PolyC binding proteins (PCBPs) impact alternative splicing of a subset of mammalian genes that are enriched in basic cellular functions. Here, we focus our analysis on PCBP-controlled cassette exon-splicing within the cell cycle control regulator cyclin-dependent kinase-2 (CDK2) transcript. We demonstrate that PCBP binding to a C-rich polypyrimidine tract (PPT) preceding exon 5 of the CDK2 transcript enhances cassette exon inclusion. This splice enhancement is U2AF65-independent and predominantly reflects actions of the PCBP1 isoform. Remarkably, PCBPs’ control of CDK2 ex5 splicing has evolved subsequent to mammalian divergence via conversion of constitutive exon 5 inclusion in the mouse CDK2 transcript to PCBP-responsive exon 5 alternative splicing in humans. Importantly, exclusion of exon 5 from the hCDK2 transcript dramatically represses the expression of CDK2 protein with a corresponding perturbation in cell cycle kinetics. These data highlight a recently evolved post-transcriptional pathway in primate species with the potential to modulate cell cycle control.

Structure of RNA Stem Loop B from the Picornavirus Replication Platform.

The presumptive RNA cloverleaf at the start of the 5'-untranslated region of the picornavirus genome is an essential element in replication. Stem loop B (SLB) of the cloverleaf is a recognition site for the host polyC-binding protein, which initiates a switch from translation to replication. Here we present the solution structure of human rhinovirus isotype 14 SLB using nuclear magnetic resonance spectroscopy. SLB adopts a predominantly A-form helical structure. The stem contains five Watson-Crick base pairs and one wobble base pair and is capped by an eight-nucleotide loop. The wobble base pair introduces perturbations into the helical parameters but does not appear to introduce flexibility. However, the helix major groove appears to be accessible. Flexibility is seen throughout the loop and in the terminal nucleotides. The pyrimidine-rich region of the loop, the apparent recognition site for the polyC-binding protein, is the most disordered region of the structure.

Identification of gamma-synuclein as a new PCBP1-interacting protein

Objectives: PolyC binding protein 1 (PCBP1) is a transcriptional regulator of human mu-opioid receptor (hMOR) gene in the CNS and is also related to cancer/diseases. It possesses multi-roles that can be mediated by protein–protein interactions. To understand the mechanism controlling PCBP1 functions, PCBP1-interacting protein was investigated.Methods: Using PCBP1 as the bait, a human brain cDNA library was screened via two-hybrid system. DNA sequence of candidate protein was confirmed using NCBI/SNP databases. Candidate protein in various cell lines was examined by RT-PCR. Glutathione-S-transferase (GST) pull-down and co-immunoprecipitation were used to validate the physical interaction. Its effects on hMOR gene regulation were examined.Results: One clone was identified as gamma-synuclein110E, an SNP of gamma-synuclein110V. The interaction between PCBP1 and gamma-synuclein110E was confirmed by further validation and GST pull-down assay. Confocal analysis showed gamma-synuclein110E mainly expressing in the cytosol of human neuronal NMB cells. This interaction was confirmed by co-immunoprecipitation with NMB lysates, containing both proteins endogenously. Ectopic expression of gamma-synuclein110E or 110V did not alter hMOR mRNA level or promoter activity, suggesting no involvement of gamma-synuclein in modulating hMOR expression. Co-immunoprecipitation using gamma-synuclein110E or 110V overexpressed NMB cells with anti-PCBP1 antibody revealed a stronger intensity of co-immunoprecipitated gamma-synuclein band using gamma-synuclein110E-overexpressed cells as compared to that using gamma-synuclein110V-overexpressed cells. Synuclein110E was also identified in H292 (lung), HT29 (colon) and T47D (breast) cells, and this physical interaction was confirmed.Conclusion: We report a newly identified PCBP1-interacting protein, gamma-synuclein110E, and provide some insight into its complex role as well as discuss potential roles of this interaction.

A novel role for poly(C) binding proteins in programmed ribosomal frameshifting.

Translational control through programmed ribosomal frameshifting (PRF) is exploited widely by viruses and increasingly documented in cellular genes. Frameshifting is induced by mRNA secondary structures that compromise ribosome fidelity during decoding of a heptanucleotide ‘slippery’ sequence. The nsp2 PRF signal of porcine reproductive and respiratory syndrome virus is distinctive in directing both −2 and −1 PRF and in its requirement for a trans-acting protein factor, the viral replicase subunit nsp1β. Here we show that the the trans-activation of frameshifting is carried out by a protein complex composed of nsp1β and a cellular poly(C) binding protein (PCBP). From the results of in vitro translation and electrophoretic mobility shift assays, we demonstrate that a PCBP/nsp1β complex binds to a C-rich sequence downstream of the slippery sequence and here mimics the activity of a structured mRNA stimulator of PRF. This is the first description of a role for a trans-acting cellular protein in PRF. The discovery broadens the repertoire of activities associated with poly(C) binding proteins and prototypes a new class of virus–host interactions.

PCBP2 Modulates Neural Apoptosis and Astrocyte Proliferation After Spinal Cord Injury.

PCBP2, a member of the poly(C)-binding protein (PCBP) family, plays a pivotal role in posttranscriptional and translational regulation by interacting with single-stranded poly(C) motifs in target mRNAs. It is reported that several PCBP family members are involved in human malignancies. However, the distribution and function of PCBP2 in the central nervous system (CNS) remain unclear. In this study, we performed an acute spinal cord injury (SCI) model in adult rats and investigated the dynamic changes of PCBP2 expression in the spinal cord. Western blot and immunohistochemistry analysis revealed that PCBP2 presented in normal spinal cord. It gradually increased, reached a peak at 3day, and then declined to basal levels at 14days after SCI. We observed that the expression of PCBP2 was enhanced in the gray and white matter. Immunofluorescence indicated that PCBP2 was located in the neurons and astrocytes. Moreover, colocalization of PCBP2/active caspase-3 was detected in neurons, and colocalization of PCBP2/proliferating cell nuclear antigen was detected in astrocytes after SCI. These results indicated that PCBP2 might play an important role in neuronal apoptosis and astrocyte proliferation. In vitro, PCBP2-specific siRNA-transfected neuron showed significantly decrease of neuronal apoptosis and expression of cell cycle related proteins following glutamate stimulation. Meanwhile, PCBP2 knockdown also reduced primary astrocytes proliferation. All above indicated that PCBP2 might play a crucial role in cell proliferation and apoptosis. Collectively, our data suggested that PCBP2 might play important roles in CNS pathophysiology after SCI.

αCP binding to a cytosine-rich subset of polypyrimidine tracts drives a novel pathway of cassette exon splicing in the mammalian transcriptome.

Alternative splicing (AS) is a robust generator of mammalian transcriptome complexity. Splice site specification is controlled by interactions of cis-acting determinants on a transcript with specific RNA binding proteins. These interactions are frequently localized to the intronic U-rich polypyrimidine tracts (PPT) located 5′ to the majority of splice acceptor junctions. αCPs (also referred to as polyC-binding proteins (PCBPs) and hnRNPEs) comprise a subset of KH-domain proteins with high affinity and specificity for C-rich polypyrimidine motifs. Here, we demonstrate that αCPs promote the splicing of a defined subset of cassette exons via binding to a C-rich subset of polypyrimidine tracts located 5′ to the αCP-enhanced exonic segments. This enhancement of splice acceptor activity is linked to interactions of αCPs with the U2 snRNP complex and may be mediated by cooperative interactions with the canonical polypyrimidine tract binding protein, U2AF65. Analysis of αCP-targeted exons predicts a substantial impact on fundamental cell functions. These findings lead us to conclude that the αCPs play a direct and global role in modulating the splicing activity and inclusion of an array of cassette exons, thus driving a novel pathway of splice site regulation within the mammalian transcriptome.

Spatiotemporal Expression of Poly(rC)-Binding Protein PCBP2 Modulates Schwann Cell Proliferation After Sciatic Nerve Injury.

Poly(C)-binding proteins (PCBPs), also known as RNA-binding proteins, interact in a sequence-specific fashion with single-stranded poly(C). It was reported that PCBP2 contributed to gastric cancer proliferation and survival through miR-34a, and knockdown of PCBP2 inhibited glioma proliferation through inhibition of cell cycle progression. In addition, PCBP2 might play a critical role in the regulation of cortical neurons apoptosis induced by hypoxia or ischemia. Because of the essential role of PCBP2 in nervous system and cell growth, we investigated the spatiotemporal expression of PCBP2 in a rat sciatic nerve crush (SNC) model. We detected the upregulated expression of PCBP2 in Schwann cell after SNC. Besides, the peak expression of PCBP2 was in parallel with proliferation cell nuclear antigen. In vitro, we observed increased expression of PCBP2 during the process of TNF-α-induced Schwann cell proliferation. Specially, PCBP2-specific siRNA-transfected Schwann cell showed significantly decreased ability for proliferation. Together, all these data indicated that the change of PCBP2 protein expression was associated with Schwann cell proliferation after the trauma of the peripheral nervous system.

(1)H, (15)N and (13)C backbone resonance assignments of the N-terminal, tandem KH domains of human hnRNP E1.

Heterogeneous nuclear ribonucleoproteins (hnRNPs) can be divided into subgroups based on their RNA-binding characteristics. One subgroup in mammalian cells are the Poly(C)-binding proteins (PCBPs) comprised of hnRNP K/J and hnRNP E1-4 [the latter also known as PCBP 1-4 or α-complex proteins (α-CP) 1-4]. Each subgroup member has three K homology (KH) nucleic acid-binding domains. Individual KH domains bind short single-stranded(ss), poly-pyrimidine-rich nucleic acid sequences with rather weak affinity. In this study, we report the (1)H, (13)C and (15)N backbone resonance assignments of the first and second KH domains of hnRNP E1, which plays a pivotal role in posttranscriptional and translational regulation of RNA targets. Our NMR assignments lay the foundation for a detailed investigation of the dynamic cooperation of the tandem KH1 and KH2 domains to bind nucleic acids.