Siew Ping Han

Functional diversity of the hnRNPs: past, present and perspectives

The hnRNPs (heterogeneous nuclear ribonucleoproteins) are RNA-binding proteins with important roles in multiple aspects of nucleic acid metabolism, including the packaging of nascent transcripts, alternative splicing and translational regulation. Although they share some general characteristics, they vary greatly in terms of their domain composition and functional properties. Although the traditional grouping of the hnRNPs as a collection of proteins provided a practical framework, which has guided much of the research on them, this approach is becoming increasingly incompatible with current knowledge about their structural and functional divergence. Hence, we review the current literature to examine hnRNP diversity, and discuss how this impacts upon approaches to the classification of RNA-binding proteins in general.

A WAVE2–Arp2/3 actin nucleator apparatus supports junctional tension at the epithelial zonula adherens

WAVE2–Arp2/3 is a major nucleator of actin assembly at the zonula adherens and likely acts in response to junctional Rac signaling. It supports myosin II recruitment to, and tension generation at, the junction.

Differential subcellular distributions and trafficking functions of hnRNP A2/B1 spliceoforms.

Trafficking of mRNA molecules from the nucleus to distal processes in neural cells is mediated by heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1 trans‐acting factors. Although hnRNP A2/B1 is alternatively spliced to generate four isoforms, most functional studies have not distinguished between these isoforms. Here, we show, using isoform‐specific antibodies and isoform‐specific green fluorescent protein (GFP)‐fusion expression constructs, that A2b is the predominant cytoplasmic isoform in neural cells, suggesting that it may play a key role in mRNA trafficking. The differential subcellular distribution patterns of the individual isoforms are determined by the presence or absence of alternative exons that also affect their dynamic behavior in different cellular compartments, as measured by fluorescence correlation spectroscopy. Expression of A2b is also differentially regulated with age, species and cellular development. Furthermore, coinjection of isoform‐specific antibodies and labeled RNA into live oligodendrocytes shows that the assembly of RNA granules is impaired by blockade of A2b function. These findings suggest that neural cells modulate mRNA trafficking by regulating alternative splicing of hnRNP A2/B1 and controlling expression levels of A2b, which may be the predominant mediator of cytoplasmic‐trafficking functions. These findings highlight the importance of considering isoform‐specific functions for alternatively spliced proteins.

Cortactin scaffolds Arp2/3 and WAVE2 at the epithelial zonula adherens.

Background: Productive epithelial interactions require actin filament assembly at E-cadherin adhesions. Results: Cortactin localizes to the zonula adherens through interactions with E-cadherin and N-WASP; there it recruits Arp2/3 and WAVE2 by separate mechanisms to support actin nucleation. Conclusion: Cortactin acts as a coincident scaffold. Significance: Cortactin can regulate the dynamic integration of cadherin adhesion with the actin cytoskeleton. Cadherin junctions arise from the integrated action of cell adhesion, signaling, and the cytoskeleton. At the zonula adherens (ZA), a WAVE2-Arp2/3 actin nucleation apparatus is necessary for junctional tension and integrity. But how this is coordinated with cadherin adhesion is not known. We now identify cortactin as a key scaffold for actin regulation at the ZA, which localizes to the ZA through influences from both E-cadherin and N-WASP. Using cell-free protein expression and fluorescent single molecule coincidence assays, we demonstrate that cortactin binds directly to the cadherin cytoplasmic tail. However, its concentration with cadherin at the apical ZA also requires N-WASP. Cortactin is known to bind Arp2/3 directly (Weed, S. A., Karginov, A. V., Schafer, D. A., Weaver, A. M., Kinley, A. W., Cooper, J. A., and Parsons, J. T. (2000) J. Cell Biol. 151, 29–40). We further show that cortactin can directly bind WAVE2, as well as Arp2/3, and both these interactions are necessary for actin assembly at the ZA. We propose that cortactin serves as a platform that integrates regulators of junctional actin assembly at the ZA.

Coronin 1B Reorganizes the Architecture of F-Actin Networks for Contractility at Steady-State and Apoptotic Adherens Junctions

In this study we sought to identify how contractility at adherens junctions influences apoptotic cell extrusion. We first found that the generation of effective contractility at steady-state junctions entails a process of architectural reorganization whereby filaments that are initially generated as poorly organized networks of short bundles are then converted into co-aligned perijunctional bundles. Reorganization requires coronin 1B, which is recruited to junctions by E-cadherin adhesion and is necessary to establish contractile tension at the zonula adherens. When cells undergo apoptosis within an epithelial monolayer, coronin 1B is also recruited to the junctional cortex at the apoptotic/neighbor cell interface in an E-cadherin-dependent fashion to support actin architectural reorganization, contractility, and extrusion. We propose that contractile stress transmitted from the apoptotic cell through E-cadherin adhesions elicits a mechanosensitive response in neighbor cells that is necessary for the morphogenetic event of apoptotic extrusion to occur.

Functional implications of the emergence of alternative splicing in hnRNP A/B transcripts

The heterogeneous nuclear ribonucleoproteins (hnRNPs) A/B are a family of RNA-binding proteins that participate in various aspects of nucleic acid metabolism, including mRNA trafficking, telomere maintenance, and splicing. They are both regulators and targets of alternative splicing, and the patterns of alternative splicing of their transcripts have diverged between paralogs and between orthologs in different species. Surprisingly, the extent of this splicing variation and its implications for post-transcriptional regulation have remained largely unexplored. Here, we conducted a detailed analysis of hnRNP A/B sequences and expression patterns across six vertebrates. Alternative exons emerged via the introduction of new splice sites, changes in the strengths of existing splice sites, and the accumulation of auxiliary splicing regulatory motifs. Observed isoform expression patterns could be attributed to the frequency and strength of cis-elements. We found a trend toward increased splicing variation in mammals and identified novel alternatively spliced isoforms in human and chicken. Pulldown and translational assays demonstrated that the inclusion of alternative exons altered the affinity of hnRNP A/B proteins for their cognate nucleic acids and modified protein expression levels. As the hnRNPs A/B regulate several key steps in mRNA processing, the involvement of diverse hnRNP isoforms in multiple cellular contexts and species implies concomitant differences in the transcriptional output of these systems. We conclude that the emergence of alternative splicing in the hnRNPs A/B has contributed to the diversification of their roles in the regulation of alternative splicing and has thus added an unexpected layer of regulatory complexity to transcription in vertebrates.

RPTPα controls epithelial adherens junctions, linking E-cadherin engagement to c-Src-mediated phosphorylation of cortactin

ABSTRACT Epithelial junctions are fundamental determinants of tissue organization, subject to regulation by tyrosine phosphorylation. Homophilic binding of E-cadherin activates tyrosine kinases, such as Src, that control junctional integrity. Protein tyrosine phosphatases (PTPs) also contribute to cadherin-based adhesion and signaling, but little is known about their specific identity or functions at epithelial junctions. Here, we report that the receptor PTP RPTP&agr; (human gene name PTPRA) is recruited to epithelial adherens junctions at the time of cell–cell contact, where it is in molecular proximity to E-cadherin. RPTP&agr; is required for appropriate cadherin-dependent adhesion and for cyst architecture in three-dimensional culture. Loss of RPTP&agr; impairs adherens junction integrity, as manifested by defective E-cadherin accumulation and peri-junctional F-actin density. These effects correlate with a role for RPTP&agr; in cellular (c)-Src activation at sites of E-cadherin engagement. Mechanistically, RPTP&agr; is required for appropriate tyrosine phosphorylation of cortactin, a major Src substrate and a cytoskeletal actin organizer. Expression of a phosphomimetic cortactin mutant in RPTP&agr;-depleted cells partially rescues F-actin and E-cadherin accumulation at intercellular contacts. These findings indicate that RPTP&agr; controls cadherin-mediated signaling by linking homophilic E-cadherin engagement to cortactin tyrosine phosphorylation through c-Src.

The cytoskeleton and classical cadherin adhesions

This chapter discusses the biochemical and functional links between classical cadherin adhesion systems and the cytoskeleton. Cadherins are best understood to cooperate with the actin cytoskeleton, but there is increasing evidence for the role of junctional microtubules in regulating cadherin biology. Cadherin adhesions and the junctional cytoskeleton are both highly dynamic systems that undergo continual assembly, turnover and remodeling, and yet maintain steady state structures necessary for intercellular adhesion. This requires the functional coordination of cadherins and cadherin-binding proteins, actin regulatory proteins, organizers of microtubule assembly and structure, and signaling pathways. These components act in concert to regulate junctional organization in response to extracellular forces and changing cellular contexts, which is essential for intercellular cohesion and tissue integrity.

A visual framework for sequence analysis using n-grams and spectral rearrangement

MOTIVATION Protein sequences are often composed of regions that have distinct evolutionary histories as a consequence of domain shuffling, recombination or gene conversion. New approaches are required to discover, visualize and analyze these sequence regions and thus enable a better understanding of protein evolution. RESULTS Here, we have developed an alignment-free and visual approach to analyze sequence relationships. We use the number of shared n-grams between sequences as a measure of sequence similarity and rearrange the resulting affinity matrix applying a spectral technique. Heat maps of the affinity matrix are employed to identify and visualize clusters of related sequences or outliers, while n-gram-based dot plots and conservation profiles allow detailed analysis of similarities among selected sequences. Using this approach, we have identified signatures of domain shuffling in an otherwise poorly characterized family, and homology clusters in another. We conclude that this approach may be generally useful as a framework to analyze related, but highly divergent protein sequences. It is particularly useful as a fast method to study sequence relationships prior to much more time-consuming multiple sequence alignment and phylogenetic analysis. AVAILABILITY A software implementation (MOSAIC) of the framework described here can be downloaded from http://bioinformatics.org.au/mosaic/ CONTACT m.ragan@uq.edu.au SUPPLEMENTARY INFORMATION Supplementary data are available at Bioinformatics online.

Differential subnuclear localisation of hnRNPs A/B is dependent on transcription and cell cycle stage.

The heterogeneous nuclear ribonucleoproteins A1, A2/B1 and A3 (hnRNPs A/B) are involved in many nuclear functions that are confined to distinct regions within the nucleus. To characterise and compare the distribution of the hnRNPs A/B in these subnuclear compartments, their colocalisation with spliceosomal components, nascent transcripts and other nuclear markers in HeLa cells was investigated by immunostaining and transfection of GFP constructs. The mechanisms of this localisation were further explored by treating cells with detergent, nucleases and transcription inhibitors. We have also examined the dynamics of A2/B1 throughout the cell cycle. Our results show that hnRNPs A/B have different subnuclear localisations, with A1 differentially localised to the nuclear envelope, and A2/B1 and A3 enriched around nucleoli. This pattern of distribution was dependent on RNA integrity and active transcription. The hnRNPs A/B preferentially colocalised with a subset of splicing factors. Significantly, only rarely did transcription factories colocalise with high levels of these hnRNPs. Moreover, localisation of A2/B1 changed with cell cycle stage. Our findings show that the subnuclear localisation of the hnRNPs A/B is differentially, spatially and temporally regulated, and suggest that this localisation may be relevant to their nuclear functions.