MiRNAs differentially expressed in vegetative and reproductive organs of Marchantia polymorpha - insights into their expression pattern, gene structures and function.

MicroRNAs regulate gene expression affecting a variety of plant developmental processes. The evolutionary position of Marchantia polymorpha makes it a significant model to understand miRNA-mediated gene regulatory pathways in plants. Previous studies focused on conserved miRNA-target mRNA modules showed their critical role in Marchantia development. Here, we demonstrate that the differential expression of conserved miRNAs among land plants and their targets in selected organs of Marchantia additionally underlines their role in regulating fundamental developmental processes. The main aim of this study was to characterize selected liverwort-specific miRNAs, as there is a limited knowledge on their biogenesis, accumulation, targets, and function in Marchantia. We demonstrate their differential accumulation in vegetative and generative organs. We reveal that all liverwort-specific miRNAs examined are encoded by independent transcriptional units. MpmiR11737a, MpmiR11887 and MpmiR11796, annotated as being encoded within protein-encoding genes, have their own independent transcription start sites. The analysis of selected liverwort-specific miRNAs and their pri-miRNAs often reveal correlation in their levels, suggesting transcriptional regulation. However, MpmiR11796 shows a reverse correlation to its pri-miRNA level, suggesting post-transcriptional regulation. Moreover, we identify novel targets for selected liverwort-specific miRNAs and demonstrate an inverse correlation between their expression and miRNA accumulation. In the case of one miRNA precursor, we provide evidence that it encodes two functional miRNAs with two independent targets. Overall, our research sheds light on liverwort-specific miRNA gene structure, provides new data on their biogenesis and expression regulation. Furthermore, identifying their targets, we hypothesize the potential role of these miRNAs in early land plant development and functioning.

tRNA-Cys gene clusters exhibit high variability in Arabidopsis thaliana.

Although most of the genes encoding tRNAs in plants are dispersed throughout the genome, a fraction of them form tRNA gene clusters. In Arabidopsis thaliana, the smallest of tRNA clusters on chromosome 5 consists of four tRNA-Cys-GCA genes placed within repeating units of 0.4 kbp. A systematic analysis of the genomic sequences of syntenic regions from various ecotypes of A. thaliana showed that the general structure of the cluster, consisting of a tRNA-Cys pseudogene followed by repeating units containing tRNA-Cys genes, is well conserved. However, there is significant heterogeneity in the number of repeating units between different ecotypes. A unique feature of this cluster is the presence of putative transposable elements (Helitron). In addition, two further tRNA-Cys gene mini-clusters (gene pairs) in A. thaliana were identified. RNA-seq-based evaluation of expression of tRNA-Cys-GCA genes showed a positive signal for 11 out of 13 unique transcripts. An analysis of the conservation of the tRNA-Cys clusters from A. thaliana with the corresponding regions from four other Arabidopsis species suggests a sequence of events that led to the divergence of these regions.

AthCNV: A Map of DNA Copy Number Variations in the Arabidopsis Genome.

Copy number variations (CNVs) greatly contribute to intraspecies genetic polymorphism and phenotypic diversity. Recent analyses of sequencing data for >1000 Arabidopsis (Arabidopsis thaliana) accessions focused on small variations and did not include CNVs. Here, we performed genome-wide analysis and identified large indels (50 to 499 bp) and CNVs (500 bp and larger) in these accessions. The CNVs fully overlap with 18.3% of protein-coding genes, with enrichment for evolutionarily young genes and genes involved in stress and defense. By combining analysis of both genes and transposable elements (TEs) affected by CNVs, we revealed that the variation statuses of genes and TEs are tightly linked and jointly contribute to the unequal distribution of these elements in the genome. We also determined the gene copy numbers in a set of 1060 accessions and experimentally validated the accuracy of our predictions by multiplex ligation-dependent probe amplification assays. We then successfully used the CNVs as markers to analyze population structure and migration patterns. Finally, we examined the impact of gene dosage variation triggered by a CNV spanning the SEC10 gene on SEC10 expression at both the transcript and protein levels. The catalog of CNVs, CNV-overlapping genes, and their genotypes in a top model dicot will stimulate the exploration of the genetic basis of phenotypic variation.

PEP444c encoded within the MIR444c gene regulates microRNA444c accumulation in barley.

MicroRNAs are small, noncoding RNA molecules that regulate the expression of their target genes. The MIR444 gene family is present exclusively in monocotyledons, and microRNAs444 from this family have been shown to target certain MADS-box transcription factors in rice and barley. We identified three barley MIR444 (MIR444a/b/c) genes and comprehensively characterised their structure and the processing pattern of the primary transcripts (pri-miRNAs444). Pri-microRNAs444 undergo extensive alternative splicing, generating functional and nonfunctional pri-miRNA444 isoforms. We show that barley pri-miRNAs444 contain numerous open reading frames (ORFs) whose transcripts associate with ribosomes. Using specific antibodies, we provide evidence that selected ORFs encoding PEP444a within MIR444a and PEP444c within MIR444c are expressed in barley plants. Moreover, we demonstrate that CRISPR-associated endonuclease 9 (Cas9)-mediated mutagenesis of the PEP444c-encoding sequence results in a decreased level of PEP444 transcript in barley shoots and roots and a 5-fold reduced level of mature microRNA444c in roots. Our observations suggest that PEP444c encoded by the MIR444c gene is involved in microRNA444c biogenesis in barley.

Biogenesis, conservation, and function of miRNA in liverworts.

MicroRNAs (miRNAs) are small non-coding endogenous RNA molecules, 18-24 nucleotides long, that control multiple gene regulatory pathways via post-transcriptional gene silencing in eukaryotes. To develop a comprehensive picture of the evolutionary history of miRNA biogenesis and action in land plants, studies on bryophyte representatives are needed. Here, we review current understanding of liverwort MIR gene structure, miRNA biogenesis, and function, focusing on the simple thalloid Pellia endiviifolia and the complex thalloid Marchantia polymorpha. We review what is known about conserved and non-conserved miRNAs, their targets, and the functional implications of miRNA action in M. polymorpha and P. endiviifolia. We note that most M. polymorpha miRNAs are encoded within protein-coding genes and provide data for 23 MIR gene structures recognized as independent transcriptional units. We identify M. polymorpha genes involved in miRNA biogenesis that are homologous to those identified in higher plants, including those encoding core microprocessor components and other auxiliary and regulatory proteins that influence the stability, folding, and processing of pri-miRNAs. We analyzed miRNA biogenesis proteins and found similar domain architecture in most cases. Our data support the hypothesis that almost all miRNA biogenesis factors in higher plants are also present in liverworts, suggesting that they emerged early during land plant evolution.

Taxonomy-aware, sequence similarity ranking reliably predicts phage-host relationships.

BACKGROUND: Characterizing phage-host interactions is critical to understanding the ecological role of both partners and effective isolation of phage therapeuticals. Unfortunately, experimental methods for studying these interactions are markedly slow, low-throughput, and unsuitable for phages or hosts difficult to maintain in laboratory conditions. Therefore, a number of in silico methods emerged to predict prokaryotic hosts based on viral sequences. One of the leading approaches is the application of the BLAST tool that searches for local similarities between viral and microbial genomes. However, this prediction method has three major limitations: (i) top-scoring sequences do not always point to the actual host; (ii) mosaic virus genomes may match to many, typically related, bacteria; and (iii) viral and host sequences may diverge beyond the point where their relationship can be detected by a BLAST alignment. RESULTS: We created an extension to BLAST, named Phirbo, that improves host prediction quality beyond what is obtainable from standard BLAST searches. The tool harnesses information concerning sequence similarity and bacteria relatedness to predict phage-host interactions. Phirbo was evaluated on three benchmark sets of known virus-host pairs, and it improved precision and recall by 11-40 percentage points over currently available, state-of-the-art, alignment-based, alignment-free, and machine-learning host prediction tools. Moreover, the discriminatory power of Phirbo for the recognition of virus-host relationships surpassed the results of other tools by at least 10 percentage points (area under the curve = 0.95), yielding a mean host prediction accuracy of 57% and 68% at the genus and family levels, respectively, and drops by 12 percentage points when using only a fraction of viral genome sequences (3 kb). Finally, we provide insights into a repertoire of protein and ncRNA genes that are shared between phages and hosts and may be prone to horizontal transfer during infection. CONCLUSIONS: Our results suggest that Phirbo is a simple and effective tool for predicting phage-host relationships.

Pi-starvation induced transcriptional changes in barley revealed by a comprehensive RNA-Seq and degradome analyses.

BACKGROUND: Small RNAs (sRNAs) are 20-30 nt regulatory elements which are responsible for plant development regulation and participate in many plant stress responses. Insufficient inorganic phosphate (Pi) concentration triggers plant responses to balance the internal Pi level. RESULTS: In this study, we describe Pi-starvation-responsive small RNAs and transcriptome changes in barley (Hordeum vulgare L.) using Next-Generation Sequencing (NGS) RNA-Seq data derived from three different types of NGS libraries: (i) small RNAs, (ii) degraded RNAs, and (iii) functional mRNAs. We find that differentially and significantly expressed miRNAs (DEMs, Bonferroni adjusted p-value < 0.05) are represented by 15 molecules in shoot and 13 in root; mainly various miR399 and miR827 isomiRs. The remaining small RNAs (i.e., those without perfect match to reference sequences deposited in miRBase) are considered as differentially expressed other sRNAs (DESs, p-value Bonferroni correction < 0.05). In roots, a more abundant and diverse set of other sRNAs (DESs, 1796 unique sequences, 0.13% from the average of the unique small RNA expressed under low-Pi) contributes more to the compensation of low-Pi stress than that in shoots (DESs, 199 unique sequences, 0.01%). More than 80% of differentially expressed other sRNAs are up-regulated in both organs. Additionally, in barley shoots, up-regulation of small RNAs is accompanied by strong induction of two nucleases (S1/P1 endonuclease and 3'-5' exonuclease). This suggests that most small RNAs may be generated upon nucleolytic cleavage to increase the internal Pi pool. Transcriptomic profiling of Pi-starved barley shoots identifies 98 differentially expressed genes (DEGs). A majority of the DEGs possess characteristic Pi-responsive cis-regulatory elements (P1BS and/or PHO element), located mostly in the proximal promoter regions. GO analysis shows that the discovered DEGs primarily alter plant defense, plant stress response, nutrient mobilization, or pathways involved in the gathering and recycling of phosphorus from organic pools. CONCLUSIONS: Our results provide comprehensive data to demonstrate complex responses at the RNA level in barley to maintain Pi homeostasis and indicate that barley adapts to Pi-starvation through elicitation of RNA degradation. Novel P-responsive genes were selected as putative candidates to overcome low-Pi stress in barley plants.

The identification of differentially expressed genes in male and female gametophytes of simple thalloid liverwort Pellia endiviifolia sp. B using an RNA-seq approach.

MAIN CONCLUSION: This study shows differences in gene expression between male and female gametophytes of the simple thalloid liverwort with a distinction between the vegetative and reproductive phases of growth. Pellia endiviifolia is a simple thalloid liverwort that, together with hornworts and mosses, represents the oldest living land plants. The limited taxon sampling for genomic and functional studies hampers our understanding of processes governing evolution of these plants. RNA sequencing represents an attractive way to elucidate the molecular mechanisms of non-model species development. In the present study, RNA-seq was used to profile the differences in gene expression between P. endiviifolia male and female gametophytes, with a distinction between the vegetative and reproductive phases of growth. By comparison of the gene expression profiles from individuals producing sex organs with the remaining thalli types, we have determined a set of genes whose expression might be important for the development of P. endiviifolia reproductive organs. The selected differentially expressed genes (DEGs) were categorized into five main pathways: metabolism, genetic information processing, environmental information processing, cellular processes, and organismal systems. A comparison of the obtained data with the Marchantia polymorpha transcriptome resulted in the identification of genes exhibiting a similar expression pattern during the reproductive phase of growth between members of the two distinct liverwort classes. The common expression profile of  87 selected genes suggests a common mechanism governing sex organ development in both liverwort species. The obtained RNA-seq results were confirmed by RT-qPCR for the DEGs with the highest differences in expression level. Five Pellia-female-specific and two Pellia-male-specific DEGs showed enriched expression in archegonia and antheridia, respectively. The identified genes are promising candidates for functional studies of their involvement in liverwort sexual reproduction.

Dual Role of the Histone Variant H2A.Z in Transcriptional Regulation of Stress-Response Genes.

The influence of the histone variant H2A.Z on transcription remains a long-standing conundrum. Here, by analyzing the actin-related protein6 mutant, which is impaired in H2A.Z deposition, and by H2A.Z profiling in stress conditions, we investigated the impact of this histone variant on gene expression in Arabidopsis thaliana We demonstrate that the arp6 mutant exhibits anomalies in response to osmotic stress. Indeed, stress-responsive genes are overrepresented among those hyperactive in arp6. In wild-type plants, these genes exhibit high levels of H2A.Z in the gene body. Furthermore, we observed that in drought-responsive genes, levels of H2A.Z in the gene body correlate with transcript levels. H2A.Z occupancy, but not distribution, changes in parallel with transcriptional changes. In particular, we observed H2A.Z loss upon transcriptional activation and H2A.Z gain upon repression. These data suggest that H2A.Z has a repressive role in transcription and counteracts unwanted expression in noninductive conditions. However, reduced activity of some genes in arp6 is associated with distinct behavior of H2A.Z at their +1 nucleosome, which exemplifies the requirement of this histone for transcription. Our data support a model where H2A.Z in gene bodies has a strong repressive effect on transcription, whereas in +1 nucleosomes, it is important for maintaining the activity of some genes.

NERD, a plant-specific GW protein, defines an additional RNAi-dependent chromatin-based pathway in Arabidopsis.

In Arabidopsis, transcriptional gene silencing (TGS) can be triggered by 24 nt small-interfering RNAs (siRNAs) through the RNA-directed DNA methylation (RdDM) pathway. By functional analysis of NERD, a GW repeat- and PHD finger-containing protein, we demonstrate that Arabidopsis harbors a second siRNA-dependent DNA methylation pathway targeting a subset of nonconserved genomic loci. The activity of the NERD-dependent pathway differs from RdDM by the fact that it relies both on silencing-related factors previously implicated only in posttranscriptional gene silencing (PTGS), including RNA-DEPENDENT RNA POLYMERASE1/6 and ARGONAUTE2, and most likely on 21 nt siRNAs. A central role for NERD in integrating RNA silencing and chromatin signals in transcriptional silencing is supported by data showing that it binds both to histone H3 and AGO2 proteins and contributes to siRNA accumulation at a NERD-targeted locus. Our results unravel the existence of a conserved chromatin-based RNA silencing pathway encompassing both PTGS and TGS components in plants.

Efficiency of PacBio long read correction by 2nd generation Illumina sequencing.

Long sequencing reads offer unprecedented opportunities in analysis and reconstruction of complex genomic regions. However, the gain in sequence length is often traded for quality. Therefore, recently several approaches have been proposed (e.g. higher sequencing coverage, hybrid assembly or sequence correction) to enhance the quality of long sequencing reads. A simple and cost-effective approach includes use of the high quality 2nd generation sequencing data to improve the quality of long reads. We designed a dedicated testing procedure and selected universal programs for long read correction, which provide as the output sequences that can be used in further genomic and transcriptomic studies. Our results show that HALC is the best choice for correction of long PacBio reads, when both, read size and quality, are the main focus of the analysis. However, the tested tools show some unexpected behaviors, including read trimming and fragmentation.

Silencing of HvGSK1.1-A GSK3/SHAGGY-Like Kinase-Enhances Barley (Hordeum vulgare L.) Growth in Normal and in Salt Stress Conditions.

Glycogen synthase kinase 3 (GSK3) is a highly conserved kinase present in all eukaryotes and functions as a key regulator of a wide range of physiological and developmental processes. The kinase, known in land plants as GSK3/SHAGGY-like kinase (GSK), is a key player in the brassinosteroid (BR) signaling pathway. The GSK genes, through the BRs, affect diverse developmental processes and modulate responses to environmental factors. In this work, we describe functional analysis of HvGSK1.1, which is one of the GSK3/SHAGGY-like orthologs in barley. The RNAi-mediated silencing of the target HvGSK1.1 gene was associated with modified expression of its paralogs HvGSK1.2, HvGSK2.1, HvGSK3.1, and HvGSK4.1 in plants grown in normal and in salt stress conditions. Low nucleotide similarity between the silencing fragment and barley GSK genes and the presence of BR-dependent transcription factors' binding sites in promoter regions of barley and rice GSK genes imply an innate mechanism responsible for co-regulation of the genes. The results of the leaf inclination assay indicated that silencing of HvGSK1.1 and the changes of GSK paralogs enhanced the BR-dependent signaling in the plants. The strongest phenotype of transgenic lines with downregulated HvGSK1.1 and GSK paralogs had greater biomass of the seedlings grown in normal conditions and salt stress as well as elevated kernel weight of plants grown in normal conditions. Both traits showed a strong negative correlation with the transcript level of the target gene and the paralogs. The characteristics of barley lines with silenced expression of HvGSK1.1 are compatible with the expected phenotypes of plants with enhanced BR signaling. The results show that manipulation of the GSK-encoding genes provides data to explore their biological functions and confirm it as a feasible strategy to generate plants with improved agricultural traits.

Defective XRN3-mediated transcription termination in Arabidopsis affects the expression of protein-coding genes.

Arabidopsis thaliana contains two nuclear XRN2/3 5'-3' exonucleases that are homologs of yeast and human Rat1/Xrn2 proteins involved in the processing and degradation of several classes of nuclear RNAs and in transcription termination of RNA polymerase II. Using strand-specific short read sequencing we show that knockdown of XRN3 leads to an altered expression of hundreds of genes and the accumulation of uncapped and polyadenylated read-through transcripts generated by inefficiently terminated Pol II. Our data support the notion that XRN3-mediated changes in the expression of a subset of genes are caused by upstream read-through transcription and these effects are enhanced by RNA-mRNA chimeras generated in xrn3 plants. In turn, read-through transcripts that are antisense to downstream genes may trigger production of siRNA. Our results highlight the importance of XRN3 exoribonuclease in Pol II transcription termination in plants and show that disturbance in this process may significantly alter gene expression.

RNAcentral: a comprehensive database of non-coding RNA sequences.

RNAcentral is a database of non-coding RNA (ncRNA) sequences that aggregates data from specialised ncRNA resources and provides a single entry point for accessing ncRNA sequences of all ncRNA types from all organisms. Since its launch in 2014, RNAcentral has integrated twelve new resources, taking the total number of collaborating database to 22, and began importing new types of data, such as modified nucleotides from MODOMICS and PDB. We created new species-specific identifiers that refer to unique RNA sequences within a context of single species. The website has been subject to continuous improvements focusing on text and sequence similarity searches as well as genome browsing functionality. All RNAcentral data is provided for free and is available for browsing, bulk downloads, and programmatic access at http://rnacentral.org/.

tRex: A Web Portal for Exploration of tRNA-Derived Fragments in Arabidopsis thaliana.

tRNA-derived fragments (tRFs) constitute a new class of short regulatory RNAs that are a product of nascent or mature tRNA processing. tRF sequences have been identified in all domains of life; however, most published research pertains to human, yeast and some bacterial organisms. Despite growing interest in plant tRFs and accumulating evidence of their function in plant development and stress responses, no public, web-based repository dedicated to these molecules is currently available. Here, we introduce tRex (http://combio.pl/trex)-the first comprehensive data-driven online resource specifically dedicated to tRFs in the model plant Arabidopsis thaliana. The portal is based on verified Arabidopsis tRNA annotation and includes in-house-generated and publicly available small RNA sequencing experiments from various tissues, ecotypes, genotypes and stress conditions. The provided web-based tools are designed in a user-friendly manner and allow for seamless exploration of the data that are presented in the form of dynamic tables and cumulative coverage profiles. The tRex database is connected to external genomic and citation resources, which makes it a one-stop solution for Arabidopsis tRF-related research.

Genomewide identification of genes involved in the potato response to drought indicates functional evolutionary conservation with Arabidopsis plants.

Potato is one of the four most important food crop plants worldwide and is strongly affected by drought. The following two pairs of potato cultivars, which are related in ancestry but show different drought tolerances, were chosen for comparative gene expression studies: Gwiazda/Oberon and Tajfun/Owacja. Comparative RNA-seq analyses of gene expression differences in the transcriptomes obtained from drought-tolerant versus drought-sensitive plants during water shortage conditions were performed. The 23 top-ranking genes were selected, 22 of which are described here as novel potato drought-responsive genes. Moreover, all but one of the potato genes selected have homologues in the Arabidopsis genome. Of the seven tested A. thaliana mutants with altered expression of the selected homologous genes, compared to the wild-type Arabidopsis plants, six showed an improved tolerance to drought. These genes encode carbohydrate transporter, mitogen-activated protein kinase kinase kinase 15 (MAPKKK15), serine carboxypeptidase-like 19 protein (SCPL19), armadillo/beta-catenin-like repeat-containing protein, high-affinity nitrate transporter 2.7 and nonspecific lipid transfer protein type 2 (nsLPT). The evolutionary conservation of the functions of the selected genes in the plant response to drought confirms the importance of these identified potato genes in the ability of plants to cope with water shortage conditions. Knowledge regarding these gene functions can be used to generate potato cultivars that are resistant to unfavourable conditions. The approach used in this work and the obtained results allowed for the identification of new players in the plant response to drought.

ORCAN-a web-based meta-server for real-time detection and functional annotation of orthologs.

Summary: ORCAN (ORtholog sCANner) is a web-based meta-server for one-click evolutionary and functional annotation of protein sequences. The server combines information from the most popular orthology-prediction resources, including four tools and four online databases. Functional annotation utilizes five additional comparisons between the query and identified homologs, including: sequence similarity, protein domain architectures, functional motifs, Gene Ontology term assignments and a list of associated articles. Furthermore, the server uses a plurality-based rating system to evaluate the orthology relationships and to rank the reference proteins by their evolutionary and functional relevance to the query. Using a dataset of ∼1 million true yeast orthologs as a sample reference set, we show that combining multiple orthology-prediction tools in ORCAN increases the sensitivity and precision by 1-2 percent points. Availability and Implementation: The service is available for free at http://www.combio.pl/orcan/ . Contact: wmk@amu.edu.pl. Supplementary information: Supplementary data are available at Bioinformatics online.

A stable tRNA-like molecule is generated from the long noncoding RNA GUT15 in Arabidopsis.

The Arabidopsis GUT15 RNA belongs to a class of noncoding RNAs that are expressed from the intergenic regions of protein-coding genes. We show that the RNA polymerase II transcribed GUT15 transcript serves as a precursor for two stable RNA species, a tRNA-like molecule and GUT15-tRF-F5, which are both encoded by the final intron in the GUT15 gene. The GUT15-encoded tRNA-like molecule cannot be autonomously transcribed by RNA polymerase III. However, this molecule contains a CCA motif, suggesting that it may enter the tRNA maturation pathway. The GUT15-encoded tRNA-like sequence has an inhibiting effect on the splicing of its host intron. Moreover, we demonstrate that the canonical tRNA genes nested within introns do not affect the splicing patterns of their host protein-coding transcripts.

5SRNAdb: an information resource for 5S ribosomal RNAs.

Ribosomal 5S RNA (5S rRNA) is the ubiquitous RNA component found in the large subunit of ribosomes in all known organisms. Due to its small size, abundance and evolutionary conservation 5S rRNA for many years now is used as a model molecule in studies on RNA structure, RNA-protein interactions and molecular phylogeny. 5SRNAdb (http://combio.pl/5srnadb/) is the first database that provides a high quality reference set of ribosomal 5S RNAs (5S rRNA) across three domains of life. Here, we give an overview of new developments in the database and associated web tools since 2002, including updates to database content, curation processes and user web interfaces.

Nicotine affects protein complex rearrangement in Caenorhabditis elegans cells.

Nicotine may affect cell function by rearranging protein complexes. We aimed to determine nicotine-induced alterations of protein complexes in Caenorhabditis elegans (C. elegans) cells, thereby revealing links between nicotine exposure and protein complex modulation. We compared the proteomic alterations induced by low and high nicotine concentrations (0.01 mM and 1 mM) with the control (no nicotine) in vivo by using mass spectrometry (MS)-based techniques, specifically the cetyltrimethylammonium bromide (CTAB) discontinuous gel electrophoresis coupled with liquid chromatography (LC)-MS/MS and spectral counting. As a result, we identified dozens of C. elegans proteins that are present exclusively or in higher abundance in either nicotine-treated or untreated worms. Based on these results, we report a possible network that captures the key protein components of nicotine-induced protein complexes and speculate how the different protein modules relate to their distinct physiological roles. Using functional annotation of detected proteins, we hypothesize that the identified complexes can modulate the energy metabolism and level of oxidative stress. These proteins can also be involved in modulation of gene expression and may be crucial in Alzheimer's disease. The findings reported in our study reveal putative intracellular interactions of many proteins with the cytoskeleton and may contribute to the understanding of the mechanisms of nicotinic acetylcholine receptor (nAChR) signaling and trafficking in cells.