Brain transcriptome analysis of a CLN2 mouse model as a function of disease progression.

BACKGROUND: Neuronal ceroid lipofuscinoses, (NCLs or Batten disease) are a group of inherited, early onset, fatal neurodegenerative diseases associated with mutations in 13 genes. All forms of the disease are characterized by lysosomal accumulation of fluorescent storage material, as well as profound neurodegeneration, but the relationship of the various genes' function to a single biological process is not obvious. In this study, we used a well-characterized mouse model of classical late infantile NCL (cLINCL) in which the tripeptidyl peptidase 1 (Tpp1) gene is disrupted by gene targeting, resulting in loss of detectable TPP1 activity and leading to progressive neurological phenotypes including ataxia, increased motor deficiency, and early death. METHODS: In order to identify genes and pathways that may contribute to progression of the neurodegenerative process, we analyzed forebrain/midbrain and cerebellar transcriptional differences at 1, 2, 3 and 4 months of age in control and TPP1-deficient mice by global RNA-sequencing. RESULTS: Progressive neurodegenerative inflammatory responses involving microglia, astrocytes and endothelial cells were observed, accompanied by activation of leukocyte extravasation signals and upregulation of nitric oxide production and reactive oxygen species. Several astrocytic (i.e., Gfap, C4b, Osmr, Serpina3n) and microglial (i.e., Ctss, Itgb2, Itgax, Lyz2) genes were identified as strong markers for assessing disease progression as they showed increased levels of expression in vivo over time. Furthermore, transient increased expression of choroid plexus genes was observed at 2 months in the lateral and fourth ventricle, highlighting an early role for the choroid plexus and cerebrospinal fluid in the disease pathology. Based on these gene expression changes, we concluded that neuroinflammation starts, for the most part, after 2 months in the Tpp1-/- brain and that activation of microglia and astrocytes occur more rapidly in cerebellum than in the rest of the brain; confirming increased severity of inflammation in this region. CONCLUSIONS: These findings have led to a better understanding of cLINCL pathological onset and progression, which may aid in development of future therapeutic treatments for this disease.

Circulating plasma exosomes in obstructive sleep apnoea and reverse dipping blood pressure.

BACKGROUND: Obstructive sleep apnoea (OSA) increases the risk of an abnormal nondipping 24 h blood pressure profile, an independent risk factor for cardiovascular disease (CVD). We examined differential exosomal microRNA (miRNA) expression in untreated OSA patients with normal dipping blood pressure (NDBP) and reverse dipping blood pressure (RDBP), an extreme form of nondipping, to understand the mechanisms underlying nondipping blood pressure in OSA. METHODS: 46 patients (15 RDBP versus 31 NDBP) matched for OSA severity (respiratory event index 32.6±22.5 versus 32.2±18.1 events·h-1; p=0.9), age (54.8±12.9 versus 49±9.9 years; p=0.09) and body mass index (36.2±6.6 versus 34.4±6.8 kg·m-2; p=0.4) were included. Plasma exosomes were characterised by flow cytometry and functional in vitro reporter assays were conducted on cultured endothelial cells. Exosome miRNA cargo was profiled with microarrays followed by bioinformatics analyses. RESULTS: Exosomes from RDBP patients increased the permeability of endothelial cell tight junctions and adhesion molecule expression. Principal component analyses of miRNA array data showed strict separation and identification of the two groups. A restricted and validated signature of exosomal miRNAs was identified in the RDBP versus NDBP group. Their predicted target genes involved phosphatidylinositol 3-kinase-Akt (p=0.004), Ras (p=3.42E-05), Wnt (p=0.003) and hypoxia inducible factor-1 signalling (p=0.04), inflammatory mediator regulation of transient receptor potential channels (p=0.01), and several cancer-related pathways. CONCLUSIONS: Patients with RDBP have altered miRNA cargoes in circulating exosomes that invoke in vitro endothelial dysfunction. A selected number of circulating exosomal miRNAs play an important role in abnormal circadian regulation of blood pressure and may provide prognostic biomarkers of CVD risk in OSA.

CD8α+ Dendritic Cells Dictate Leukemia-Specific CD8+ T Cell Fates.

APCs are essential for the orchestration of antitumor T cell responses. Batf3-lineage CD8α+ and CD103+ dendritic cells (DCs), in particular, are required for the spontaneous initiation of CD8+ T cell priming against solid tumors. In contrast, little is known about the APCs that regulate CD8+ T cell responses against hematological malignancies. Using an unbiased approach, we aimed to characterize the APCs responsible for regulating CD8+ T cell responses in a syngeneic murine leukemia model. We show with single-cell resolution that CD8α+ DCs alone acquire and cross-present leukemia Ags in vivo, culminating in the induction of leukemia-specific CD8+ T cell tolerance. Furthermore, we demonstrate that the mere acquisition of leukemia cell cargo is associated with a unique transcriptional program that may be important in regulating tolerogenic CD8α+ DC functions in mice with leukemia. Finally, we show that systemic CD8α+ DC activation with a TLR3 agonist completely prevents their ability to generate leukemia-specific CD8+ T cell tolerance in vivo, resulting instead in the induction of potent antileukemia T cell immunity and prolonged survival of leukemia-bearing mice. Together, our data reveal that Batf3-lineage DCs imprint disparate CD8+ T cell fates in hosts with solid tumors versus systemic leukemia.

The side population enriches for leukemia-propagating cell activity and Wnt pathway expression in zebrafish acute lymphoblastic leukemia.

Cancer stem cells have been strongly linked to resistance and relapse in many malignancies. However, purifying them from within the bulk tumor has been challenging, so their precise genetic and functional characteristics are not well defined. The side population assay exploits the ability of some cells to efflux Hoechst dye via ATP-binding cassette transporters. Stem cells have increased expression of these transporters and this assay has been shown to enrich for stem cells in various tissues and cancers. This study identifies the side population within a zebrafish model of acute lymphoblastic leukemia and correlates the frequency of side population cells with the frequency of leukemia stem cells (more precisely referred to as leukemia-propagating cells within our transplantation model). In addition, the side population within the leukemia evolves with serial transplantation, increasing in tandem with leukemia-propagating cell frequency over subsequent generations. Sorted side population cells from these tumors are enriched for leukemia-propagating cells and have enhanced engraftment compared to sorted non-side population cells when transplanted into syngeneic recipients. RNA-sequencing analysis of sorted side population cells compared to non-side population cells identified a shared expression profile within the side population and pathway analysis yielded Wnt-signaling as the most overrepresented. Gene set enrichment analysis showed that stem cell differentiation and canonical Wnt-signaling were significantly upregulated in the side population. Overall, these results demonstrate that the side population in zebrafish acute lymphoblastic leukemia significantly enriches for leukemia-propagating cells and identifies the Wnt pathway as a likely genetic driver of leukemia stem cell fate.

Cohesin-mediated interactions organize chromosomal domain architecture.

To ensure proper gene regulation within constrained nuclear space, chromosomes facilitate access to transcribed regions, while compactly packaging all other information. Recent studies revealed that chromosomes are organized into megabase-scale domains that demarcate active and inactive genetic elements, suggesting that compartmentalization is important for genome function. Here, we show that very specific long-range interactions are anchored by cohesin/CTCF sites, but not cohesin-only or CTCF-only sites, to form a hierarchy of chromosomal loops. These loops demarcate topological domains and form intricate internal structures within them. Post-mitotic nuclei deficient for functional cohesin exhibit global architectural changes associated with loss of cohesin/CTCF contacts and relaxation of topological domains. Transcriptional analysis shows that this cohesin-dependent perturbation of domain organization leads to widespread gene deregulation of both cohesin-bound and non-bound genes. Our data thereby support a role for cohesin in the global organization of domain structure and suggest that domains function to stabilize the transcriptional programmes within them.

Comparative proteomic analysis of extracellular secreted proteins expressed by two pathogenic Acanthamoeba castellanii clinical isolates and a non-pathogenic ATCC strain.

Acanthamoeba keratitis (AK) is a serious ocular disease caused by pathogenic Acanthamoeba gaining entry through wounds in the corneal injury; generally, patients at risk for contracting AK wear contact lenses, usually over a long period of time. Moreover, pathogenic Acanthamoeba causes serious consequences: it makes the cornea turbid and difficult to operate on, including procedures such as enucleation of the eyeball. At present, diagnosis of this disease is not straightforward, and treatment is very demanding. We have established the comparative transcriptome and extracellular secreted proteomic database according to the non-pathogenic strain ATCC 30010 and the pathogenic strains NCKU_B and NCKU_D. We identified 44 secreted proteins successfully, 10 consensus secreted proteins and 34 strain-specific secreted proteins. These proteins may provide targets for therapy and immuno-diagnosis of Acanthamoeba infections. This study shows a suitable approach to identify secreted proteins in Acanthamoeba and provides new perspectives for the study of molecules potentially involved in the AK.

MetaMirClust: discovery of miRNA cluster patterns using a data-mining approach.

Recent genome-wide surveys on ncRNA have revealed that a substantial fraction of miRNA genes is likely to form clusters. However, the evolutionary and biological function implications of clustered miRNAs are still elusive. After identifying clustered miRNA genes under different maximum inter-miRNA distances (MIDs), this study intended to reveal evolution conservation patterns among these clustered miRNA genes in metazoan species using a computation algorithm. As examples, a total of 15-35% of known and predicted miRNA genes in nine selected species constitute clusters under the MIDs ranging from 1kb to 50kb. Intriguingly, 33 out of 37 metazoan miRNA clusters in 56 metazoan genomes are co-conserved with their up/down-stream adjacent protein-coding genes. Meanwhile, a co-expression pattern of miR-1 and miR-133a in the mir-133-1 cluster has been experimentally demonstrated. Therefore, the MetaMirClust database provides a useful bioinformatic resource for biologists to facilitate the advanced interrogations on the composition of miRNA clusters and their evolution patterns.

Aberrant expression of miR-196a in gastric cancers and correlation with recurrence.

MicroRNAs (miRNAs) are short noncoding RNAs (~22 nt) that play important roles in the pathogenesis of human diseases by negatively regulating gene expression. Here, we examined the relationship between miR-196a and gastric cancer.By the analysis of 72 gastric cancer samples, we found that the expression level of miR-196a microRNA significantly increased in primary gastric cancer tissues versus adjacent normal tissues. In addition, extracellular miR-196a detected in conditioned medium was strongly correlated with its cellular expression status and increased circulating miR-196a in patient serum was associated with gastric cancer disease status and relapse. Furthermore, ectopic expression of miR-196a microRNA promoted the epithelial-mesenchymal transition and migration/invasion capabilities of transfected cells, suggesting its oncogenic potential in gastric cancer progression. Altogether, our data demonstrate that miR-196a exerts an oncogenic role in gastric cancer and miR-196a may be a novel biomarker for detecting gastric cancer and for monitoring disease recurrence.

Transcriptional regulation of miR-196b by ETS2 in gastric cancer cells.

E26 transformation-specific sequence (ETS)-2 is a transcriptional modulator located on chromosome 21, alterations in its expression have been implicated with a reduced incidence of solid tumors in Down syndrome patients. MicroRNAs (miRNAs) are thought to participate in diverse biological functions; however, the regulation of miRNAs is not well characterized. Recently, we reported that miR-196b is highly expressed in gastric cancers. Herein, we demonstrate that miR-196b expression was significantly repressed by ETS2 during gastric cancer oncogenesis. We demonstrate that knockdown of endogenous ETS2 expression increases miR-196b expression. A genomic region between -751 and -824 bp upstream of the miR-196b transcriptional start site was found to be critical for the repression activity. This putative regulatory promoter region contains three potential ETS2-binding motifs. Mutations within the ETS2 binding sites blocked the repression activity of ETS2. Furthermore, knockdown of ETS2 or overexpression of miR-196b significantly induced migration and invasion in gastric cancer cells. In addition, alterations in ETS2 and miR-196b expression in gastric cancer cell lines affected the expression of epithelial-mesenchymal transition-related genes. The levels of vimentin, matrix metalloproteinase (MMP)-2 and MMP9 were drastically induced, but levels of E-cadherin were decreased in shETS2- or miR-196b-transfected cells. Our data indicate that ETS2 plays a key role in controlling the expression of miR-196b, and miR-196b may mediate the tumor suppressor effects of ETS2. We demonstrated that miR-196b was transcriptionally regulated by ETS2 and there was an inverse expression profile between miR-196b and ETS2 in clinical samples. This finding could be beneficial for the development of effective cancer diagnostic and alternative therapeutic strategies.

Interrogation of rabbit miRNAs and their isomiRs.

Rabbit (Oryctolagus cuniculus) is the only lagomorph animal of which the genome has been sequenced. Establishing a rabbit miRNA resource will benefit subsequent functional genomic studies in mammals. We have generated small RNA sequence reads with SOLiD and Solexa platforms to identify rabbit miRNAs, where we identified 464 pre-miRNAs and 886 mature miRNAs. The brain and heart miRNA libraries were used for further in-depth analysis of isomiR distributions. There are several intriguing findings. First, several rabbit pre-miRNAs form highly conserved clusters. Second, there is a preference in selecting one strand as mature miRNA, resulting in an arm selection preference. Third, we analyzed the isomiR expression and validated the expression of isomiR types in different rabbit tissues. Moreover, we further performed additional small RNA libraries and defined miRNAs differentially expressed between brain and heart. We conclude also that isomiR distribution profiles could vary between brain and heart tissues.

Histone H2A ubiquitination resulting from Brap loss of function connects multiple aging hallmarks and accelerates neurodegeneration.

Aging is an intricate process characterized by multiple hallmarks including stem cell exhaustion, genome instability, epigenome alteration, impaired proteostasis, and cellular senescence. Whereas each of these traits is detrimental at the cellular level, it remains unclear how they are interconnected to cause systemic organ deterioration. Here we show that abrogating Brap, a BRCA1-associated protein essential for neurogenesis, results in persistent DNA double-strand breaks and elevation of histone H2A mono- and poly-ubiquitination (H2Aub). These defects extend to cellular senescence and proteasome-mediated histone H2A proteolysis with alterations in cells' proteomic and epigenetic states. Brap deletion in the mouse brain causes neuroinflammation, impaired proteostasis, accelerated neurodegeneration, and substantially shortened the lifespan. We further show the elevation of H2Aub also occurs in human brain tissues with Alzheimer's disease. These data together suggest that chromatin aberrations mediated by H2Aub may act as a nexus of multiple aging hallmarks and promote tissue-wide degeneration.

Enterococcus faecalis promotes a migratory and invasive phenotype in colon cancer cells.

Much about the role of intestinal microbes at the site of colon cancer development and tumor progression following curative resection remains to be understood. We have recently shown that collagenolytic bacteria such as Enterococcus faecalis predominate within the colon postoperatively, particularly at the site of the colon reconnection (i.e. anastomosis) in the early period of post-surgical recovery. The presence of collagenolytic bacteria at this site correlates with the tumor progression in a mouse model of post-surgical tumor development. In the present study we hypothesized, that collagenolytic bacteria, such as E. faecalis, play an important yet to be discovered role in tumor formation and progression. Therefore the aims of this study were to assess the role of collagenolytic E. faecalis on the migration and invasion of a murine colon cancer cell line. Results demonstrated that both migration and invasion were induced by E. faecalis with collagenolytic activity being required for only invasion. Bidirectional signaling in the E. faecalis-cancer cell interaction was observed by the discovering that the expression of gelE in E. faecalis, the gene required for collagenase production, is expressed in response to exposure to CT26 cells. The mechanism by which migration enhancement via E. faecalis occurs appears to be dependent on its ability to activate pro-uPA, a key element of the urokinase-plasminogen system, a pathway that is well - known to be important in cancer cell invasion and migration. Finally, we demonstrated that collagenase producing microbes preferentially colonize human colon cancer specimens.

UMARS: Un-MAppable Reads Solution.

BACKGROUND: Un-MAppable Reads Solution (UMARS) is a user-friendly web service focusing on retrieving valuable information from sequence reads that cannot be mapped back to reference genomes. Recently, next-generation sequencing (NGS) technology has emerged as a powerful tool for generating high-throughput sequencing data and has been applied to many kinds of biological research. In a typical analysis, adaptor-trimmed NGS reads were first mapped back to reference sequences, including genomes or transcripts. However, a fraction of NGS reads failed to be mapped back to the reference sequences. Such un-mappable reads are usually imputed to sequencing errors and discarded without further consideration. METHODS: We are investigating possible biological relevance and possible sources of un-mappable reads. Therefore, we developed UMARS to scan for virus genomic fragments or exon-exon junctions of novel alternative splicing isoforms from un-mappable reads. For mapping un-mappable reads, we first collected viral genomes and sequences of exon-exon junctions. Then, we constructed UMARS pipeline as an automatic alignment interface. RESULTS: By demonstrating the results of two UMARS alignment cases, we show the applicability of UMARS. We first showed that the expected EBV genomic fragments can be detected by UMARS. Second, we also detected exon-exon junctions from un-mappable reads. Further experimental validation also ensured the authenticity of the UMARS pipeline. The UMARS service is freely available to the academic community and can be accessed via http://musk.ibms.sinica.edu.tw/UMARS/. CONCLUSIONS: In this study, we have shown that some un-mappable reads are not caused by sequencing errors. They can originate from viral infection or transcript splicing. Our UMARS pipeline provides another way to examine and recycle the un-mappable reads that are commonly discarded as garbage.

Epigenetic regulation of miR-34b and miR-129 expression in gastric cancer.

MicroRNAs (miRNAs) are small noncoding RNAs that play fundamental roles in diverse biological and pathological processes by targeting the expression of specific genes. Here, we identified 38 methylation-associated miRNAs, the expression of which could be epigenetically restored by cotreatment with 5-aza-2'-deoxycytidine and trichostatin A. Among these 38 miRNAs, we further analyzed miR-34b, miR-127-3p, miR-129-3p and miR-409 because CpG islands are predicted adjacent to them. The methylation-silenced expression of these miRNAs could be reactivated in gastric cancer cells by treatment with demethylating drugs in a time-dependent manner. Analysis of the methylation status of these miRNAs showed that the upstream CpG-rich regions of mir-34b and mir-129-2 are frequently methylated in gastric cancer tissues compared to adjacent normal tissues, and their methylation status correlated inversely with their expression patterns. The expression of miR-34b and miR-129-3p was downregulated by DNA hypermethylation in primary gastric cancers, and the low expression was associated with poor clinicopathological features. In summary, our study shows that tumor-specific methylation silences miR-34b and miR-129 in gastric cancer cells.

Identification of homologous microRNAs in 56 animal genomes.

MicroRNAs (miRNAs) are endogenous non-protein-coding RNAs of approximately 22 nucleotides. Thousands of miRNA genes have been identified (computationally and/or experimentally) in a variety of organisms, which suggests that miRNA genes have been widely shared and distributed among species. Here, we used unique miRNA sequence patterns to scan the genome sequences of 56 bilaterian animal species for locating candidate miRNAs first. The regions centered surrounding these candidate miRNAs were then extracted for folding and calculating the features of their secondary structure. Using a support vector machine (SVM) as a classifier combined with these features, we identified an additional 13,091 orthologous or paralogous candidate pre-miRNAs, as well as their corresponding candidate mature miRNAs. Stem-loop RT-PCR and deep sequencing methods were used to experimentally validate the prediction results in human, medaka and rabbit. Our prediction pipeline allows the rapid and effective discovery of homologous miRNAs in a large number of genomes.

DNA Methylation in LIME1 and SPTBN2 Genes Is Associated with Attention Deficit in Children.

DNA methylation levels are associated with neurodevelopment. Attention-deficit/hyperactivity disorder (ADHD), characterized by attention deficits, is a common neurodevelopmental disorder. We used methylation microarray and pyrosequencing to detect peripheral blood DNA methylation markers of ADHD. DNA methylation profiling data from the microarray assays identified potential differentially methylated CpG sites between 12 ADHD patients and 9 controls. Five candidate CpG sites (cg00446123, cg20513976, cg07922513, cg17096979, and cg02506324) in four genes (LIME1, KCNAB2, CAPN9, and SPTBN2) were further examined with pyrosequencing. The attention of patients were tested using the Conners' Continuous Performance Test (CPT). In total, 126 ADHD patients with a mean age of 9.2 years (78.6% males) and 72 healthy control subjects with a mean age of 9.3 years (62.5% males) were recruited. When all participants were categorized by their CPT performance, the DNA methylation levels in LIME1 (cg00446123 and cg20513976) were found to be significantly higher and those in SPTBN2 (cg02506324) were significantly lower in children with worse CPT performance. Therefore, DNA methylation of two CpG sites in LIME1 and one CpG site in SPTBN2 is associated with attention deficits in children. DNA methylation biomarkers may assist in identifying attention deficits of children in clinical settings.

Learning to predict expression efficacy of vectors in recombinant protein production.

BACKGROUND: Recombinant protein production is a useful biotechnology to produce a large quantity of highly soluble proteins. Currently, the most widely used production system is to fuse a target protein into different vectors in Escherichia coli (E. coli). However, the production efficacy of different vectors varies for different target proteins. Trial-and-error is still the common practice to find out the efficacy of a vector for a given target protein. Previous studies are limited in that they assumed that proteins would be over-expressed and focused only on the solubility of expressed proteins. In fact, many pairings of vectors and proteins result in no expression. RESULTS: In this study, we applied machine learning to train prediction models to predict whether a pairing of vector-protein will express or not express in E. coli. For expressed cases, the models further predict whether the expressed proteins would be soluble. We collected a set of real cases from the clients of our recombinant protein production core facility, where six different vectors were designed and studied. This set of cases is used in both training and evaluation of our models. We evaluate three different models based on the support vector machines (SVM) and their ensembles. Unlike many previous works, these models consider the sequence of the target protein as well as the sequence of the whole fusion vector as the features. We show that a model that classifies a case into one of the three classes (no expression, inclusion body and soluble) outperforms a model that considers the nested structure of the three classes, while a model that can take advantage of the hierarchical structure of the three classes performs slight worse but comparably to the best model. Meanwhile, compared to previous works, we show that the prediction accuracy of our best method still performs the best. Lastly, we briefly present two methods to use the trained model in the design of the recombinant protein production systems to improve the chance of high soluble protein production. CONCLUSION: In this paper, we show that a machine learning approach to the prediction of the efficacy of a vector for a target protein in a recombinant protein production system is promising and may compliment traditional knowledge-driven study of the efficacy. We will release our program to share with other labs in the public domain when this paper is published.

Discovery and characterization of medaka miRNA genes by next generation sequencing platform.

BACKGROUND: MicroRNAs (miRNAs) are endogenous non-protein-coding RNA genes which exist in a wide variety of organisms, including animals, plants, virus and even unicellular organisms. Medaka (Oryzias latipes) is a useful model organism among vertebrate animals. However, no medaka miRNAs have been investigated systematically. It is beneficial to conduct a genome-wide miRNA discovery study using the next generation sequencing (NGS) technology, which has emerged as a powerful sequencing tool for high-throughput analysis. RESULTS: In this study, we adopted ABI SOLiD platform to generate small RNA sequence reads from medaka tissues, followed by mapping these sequence reads back to medaka genome. The mapped genomic loci were considered as candidate miRNAs and further processed by a support vector machine (SVM) classifier. As result, we identified 599 novel medaka pre-miRNAs, many of which were found to encode more than one isomiRs. Besides, additional minor miRNAs (also called miRNA star) can be also detected with the improvement of sequencing depth. These quantifiable isomiRs and minor miRNAs enable us to further characterize medaka miRNA genes in many aspects. First of all, many medaka candidate pre-miRNAs position close to each other, forming many miRNA clusters, some of which are also conserved across other vertebrate animals. Secondly, during miRNA maturation, there is an arm selection preference of mature miRNAs within precursors. We observed the differences on arm selection preference between our candidate pre-miRNAs and their orthologous ones. We classified these differences into three categories based on the distribution of NGS reads. Finally, we also investigated the relationship between conservation status and expression level of miRNA genes. We concluded that the evolutionally conserved miRNAs were usually the most abundant ones. CONCLUSIONS: Medaka is a widely used model animal and usually involved in many biomedical studies, including the ones on development biology. Identifying and characterizing medaka miRNA genes would benefit the studies using medaka as a model organism.

Sequence features involved in the mechanism of 3' splice junction wobbling.

BACKGROUND: Alternative splicing is an important mechanism mediating the diversified functions of genes in multicellular organisms, and such event occurs in around 40-60% of human genes. Recently, a new splice-junction wobbling mechanism was proposed that subtle modifications exist in mRNA maturation by alternatively choosing at 5'- GTNGT and 3'- NAGNAG, which created single amino acid insertion and deletion isoforms. RESULTS: By browsing the Alternative Splicing Database information, we observed that most 3' alternative splice site choices occur within six nucleotides of the dominant splice site and the incidence significantly decreases further away from the dominant acceptor site. Although a lower frequency of alternative splicing occurs within the intronic region (alternative splicing at the proximal AG) than in the exonic region (alternative splicing at the distal AG), alternative AG sites located within the intronic region show stronger potential as the acceptor. These observations revealed that the choice of 3' splice sites during 3' splicing junction wobbling could depend on the distance between the duplicated AG and the branch point site (BPS). Further mutagenesis experiments demonstrated that the distance of AG-to-AG and BPS-to-AG can greatly influence 3' splice site selection. Knocking down a known alternative splicing regulator, hSlu7, failed to affect wobble splicing choices. CONCLUSION: Our results implied that nucleotide distance between proximal and distal AG sites has an important regulatory function. In this study, we showed that occurrence of 3' wobble splicing occurs in a distance-dependent manner and that most of this wobble splicing is probably caused by steric hindrance from a factor bound at the neighboring tandem motif sequence.