Network-based bioinformatics analysis of spatio-temporal RNA-Seq data reveals transcriptional programs underpinning normal and aberrant retinal development.

BACKGROUND: The retina as a model system with extensive information on genes involved in development/maintenance is of great value for investigations employing deep sequencing to capture transcriptome change over time. This in turn could enable us to find patterns in gene expression across time to reveal transition in biological processes. METHODS: We developed a bioinformatics pipeline to categorize genes based on their differential expression and their alternative splicing status across time by binning genes based on their transcriptional kinetics. Genes within same bins were then leveraged to query gene annotation databases to discover molecular programs employed by the developing retina. RESULTS: Using our pipeline on RNA-Seq data obtained from fractionated (nucleus/cytoplasm) developing retina at embryonic day (E) 16 and postnatal day (P) 0, we captured high-resolution as in the difference between the cytoplasm and the nucleus at the same developmental time. We found de novo transcription of genes whose transcripts were exclusively found in the nuclear transcriptome at P0. Further analysis showed that these genes enriched for functions that are known to be executed during postnatal development, thus showing that the P0 nuclear transcriptome is temporally ahead of that of its cytoplasm. We extended our strategy to perform temporal analysis comparing P0 data to either P21-Nrl-wildtype (WT) or P21-Nrl-knockout (KO) retinae, which predicted that the KO retina would have compromised vasculature. Indeed, histological manifestation of vasodilation has been reported at a later time point (P60). CONCLUSIONS: Thus, our approach was predictive of a phenotype before it presented histologically. Our strategy can be extended to investigating the development and/or disease progression of other tissue types.

Minor splicing snRNAs are enriched in the developing mouse CNS and are crucial for survival of differentiating retinal neurons.

In eukaryotes, gene expression requires splicing, which starts with the identification of exon-intron boundaries by the small, nuclear RNA (snRNAs) of the spliceosome, aided by associated proteins. In the mammalian genome, <1% of introns lack canonical exon-intron boundary sequences and cannot be spliced by the canonical splicing machinery. These introns are spliced by the minor spliceosome, consisting of unique snRNAs (U11, U12, U4atac, and U6atac). The importance of the minor spliceosome is underscored by the disease microcephalic osteodysplastic primordial dwarfism type 1 (MOPD1), which is caused by mutation in U4atac. Thus, it is important to understand the expression and function of the minor spliceosome and its targets in mammalian development, for which we used the mouse as our model. Here, we report enrichment of the minor snRNAs in the developing head/central nervous system (CNS) between E9.5 and E12.5, along with enrichment of these snRNAs in differentiating retinal neurons. Moreover, dynamic expression kinetics of minor intron-containing genes (MIGs) was observed across retinal development. DAVID analysis of MIGs that were cotranscriptionally upregulated embryonically revealed enrichment for RNA metabolism and cell cycle regulation. In contrast, MIGs that were cotranscriptionally upregulated postnatally revealed enrichment for protein localization/transport, vesicle-mediated transport, and calcium transport. Finally, we used U12 morpholino to inactivate the minor spliceosome in the postnatal retina, which resulted in apoptosis of differentiating retinal neurons. Taken together, our data suggest that the minor spliceosome may have distinct functions in embryonic versus postnatal development. Importantly, we show that the minor spliceosome is crucial for the survival of terminally differentiating retinal neurons.

Replication-dependent histone genes are actively transcribed in differentiating and aging retinal neurons.

In the mammalian genome, each histone family contains multiple replication-dependent paralogs, which are found in clusters where their transcription is thought to be coupled to the cell cycle. Here, we wanted to interrogate the transcriptional regulation of these paralogs during retinal development and aging. We employed deep sequencing, quantitative PCR, in situ hybridization (ISH), and microarray analysis, which revealed that replication-dependent histone genes were not only transcribed in progenitor cells but also in differentiating neurons. Specifically, by ISH analysis we found that different histone genes were actively transcribed in a subset of neurons between postnatal day 7 and 14. Interestingly, within a histone family, not all paralogs were transcribed at the same level during retinal development. For example, expression of Hist1h1b was higher embryonically, while that of Hist1h1c was higher postnatally. Finally, expression of replication-dependent histone genes was also observed in the aging retina. Moreover, transcription of replication-dependent histones was independent of rapamycin-mediated mTOR pathway inactivation. Overall, our data suggest the existence of variant nucleosomes produced by the differential expression of the replication-dependent histone genes across retinal development. Also, the expression of a subset of replication-dependent histone isotypes in senescent neurons warrants re-examining these genes as "replication-dependent." Thus, our findings underscore the importance of understanding the transcriptional regulation of replication-dependent histone genes in the maintenance and functioning of neurons.

Determination of the cationic amphiphilic drug-DNA binding mode and DNA-assisted fluorescence resonance energy transfer amplification.

Understanding the mechanism of drug-DNA binding is crucial for predicting the potential genotoxicity of drugs. Agarose gel electrophoresis, absorption, steady state fluorescence, and circular dichroism have been used in exploring the interaction of cationic amphiphilic drugs (CADs) such as amitriptyline hydrochloride (AMT), imipramine hydrochloride (IMP), and promethazine hydrochloride (PMT) with calf thymus or pUC19 DNA. Agarose gel electrophoresis assay, along with absorption and steady state fluorescence studies, reveal interaction between the CADs and DNA. A comparative study of the drugs with respect to the effect of urea, iodide induced quenching, and ethidium bromide (EB) exclusion assay reflects binding of CADs to the DNA primarily in an intercalative fashion. Circular dichroism data also support the intercalative mode of binding. Besides quenching, there is fluorescence exchange energy transfer (FRET) in between CADs and EB using DNA as a template.

Expression analysis of an evolutionarily conserved alternative splicing factor, Sfrs10, in age-related macular degeneration.

Age-related macular degeneration (AMD) is the most common cause of blindness in the elderly population. Hypoxic stress created in the micro-environment of the photoreceptors is thought to be the underlying cause that results in the pathophysiology of AMD. However, association of AMD with alternative splicing mediated gene regulation is not well explored. Alternative Splicing is one of the primary mechanisms in humans by which fewer protein coding genes are able to generate a vast proteome. Here, we investigated the expression of a known stress response gene and an alternative splicing factor called Serine-Arginine rich splicing factor 10 (Sfrs10). Sfrs10 is a member of the serine-arginine (SR) rich protein family and is 100% identical at the amino acid level in most mammals. Immunoblot analysis on retinal extracts from mouse, rat, and chicken showed a single immunoreactive band. Further, immunohistochemistry on adult mouse, rat and chicken retinae showed pan-retinal expression. However, SFRS10 was not detected in normal human retina but was observed as distinct nuclear speckles in AMD retinae. This is in agreement with previous reports that show Sfrs10 to be a stress response gene, which is upregulated under hypoxia. The difference in the expression of Sfrs10 between humans and lower mammals and the upregulation of SFRS10 in AMD is further reflected in the divergence of the promoter sequence between these species. Finally, SFRS10+ speckles were independent of the SC35+ SR protein speckles or the HSF1+ stress granules. In all, our data suggests that SFRS10 is upregulated and forms distinct stress-induced speckles and might be involved in AS of stress response genes in AMD.

The expression analysis of Sfrs10 and Celf4 during mouse retinal development.

Processing of mRNAs including, alternative splicing (AS), mRNA transport and translation regulation are crucial to eukaryotic gene expression. For example, >90% of the genes in the human genome are known to undergo alternative splicing thereby expanding the proteome production capacity of a limited number of genes. Similarly, mRNA export and translation regulation plays a vital role in regulating protein production. Thus, it is important to understand how these RNA binding proteins including alternative splicing factors (ASFs) and mRNA transport and translation factors regulate these processes. Here we report the expression of an ASF, serine-arginine rich splicing factor 10 (Sfrs10) and a mRNA translation regulation factor, CUGBP, elav like family member 4 (Celf4) in the developing mouse retina. Sfrs10 was expressed throughout postnatal (P) retinal development and was observed progressively in newly differentiating neurons. Immunofluorescence (IF) showed Sfrs10 in retinal ganglion cells (RGCs) at P0, followed by amacrine and bipolar cells, and at P8 it was enriched in red/green cone photoreceptor cells. By P22, Sfrs10 was observed in rod photoreceptors in a peri-nuclear pattern. Like Sfrs10, Celf4 expression was also observed in the developing retina, but with two distinct retinal isoforms. In situ hybridization (ISH) showed progressive expression of Celf4 in differentiating neurons, which was confirmed by IF that showed a dynamic shift in Celf4 localization. Early in development Celf4 expression was restricted to the nuclei of newly differentiating RGCs and later (E16 onwards) it was observed in the initial segments of RGC axons. Later, during postnatal development, Celf4 was observed in amacrine and bipolar cells, but here it was predominantly cytoplasmic and enriched in the two synaptic layers. Specifically, at P14, Celf4 was observed in the synaptic boutons of rod bipolar cells marked by Pkc-α. Thus, Celf4 might be regulating AS early in development besides its known role of regulating mRNA localization/translation. In all, our data suggests an important role for AS and mRNA localization/translation in retinal neuron differentiation.

Computational prediction and characterisation of ubiquitously expressed new splice variant of Prkaca gene in mouse.

Prkaca gene of mouse encodes for a cAMP dependent protein kinase catalytic alpha subunit. PKA occurs naturally as a 4-membered structure having two regulatory (R) and two catalytic (C) subunits each encoded by separate gene. Alternatively spliced two transcript variants are known for the Prkaca gene, which encode for two isoforms of PKA C-subunits, namely Cα1 and Cα2. These isoforms arise as a result of alternative splicing of the first coding exon with the internal exons. We have identified a new transcript variant using combinatorial approach of bioinformatics and molecular biology techniques involving RT-PCR, semi-nested PCR and sequencing. The new transcript variant encoding Cα3 isoform has N-terminus that differs from Cα1 and Cα2 isoforms. Cα3 isoform also arise as a result of alternative splicing of first coding exon with the internal exon. Newly identified transcript is expressed ubiquitously in different tissues examined.

Alternatively spliced variants of gamma-subunit of muscle-type acetylcholine receptor in fetal and adult skeletal muscle of mouse.

Gamma-subunit of nicotinic acetylcholine receptor is encoded by chrng gene of mouse. This gene is located on chromosome 1, spans 6.5 kb, and contains 12 exons and 11 introns. Previous studies have reported three transcript variants (C1-3) produced by alternative splicing; C1 contains all the 12 reported exons, C2 uses an in-frame alternate splice site in exon-2, and C3 produced by exon-5 skipping. These variants differ in their channel kinetics and opening times. In our study, we report the presence of two new transcript variants (T1 and T2) of chrng expressed in mouse postnatal day 3 and adult skeletal muscles. These transcripts contain novel first coding exon either N1 or N2. N1 is located in the 5' UTR, while N2 is an extended exon-2. 5' extension of exon-2 contains an initiation codon which produces a novel transcript variant. Either of the two exons can splice with the internal exons to produce mature transcripts making different 5' ends of the transcripts. Consequently, the proteins encoded by these two transcripts differ at N-termini. The presence of N2 exon containing transcript was further supported by the availability of EST from the database. These new variants display heterogeneous properties. They differ in the presence of signal peptide, phosphorylation, and acetylation of their amino acid residues of the new N-termini of the gamma subunit.

Two novel N-terminal coding exons of Prkar1b gene of mouse: identified using a novel approach of in silico and molecular biology techniques.

The Prkar1b gene encodes regulatory type 1 beta subunit of protein kinase A. Here we report that mouse R1ß gene produces three alternative splice variants (designated as mR1ß1, mR1ß2 and mR1ß3) that have different N-terminal protein structures. New splice variants were identified using combinatorial approach of bioinformatics pipeline involving online available tools and databases, and molecular biology techniques involving RT-PCR, semi-nested PCR and sequencing. Except mR1ß3, which was not detected by RT-PCR in brain and muscle tissues of 3day old mice, all three spliced isoforms were found to be ubiquitously expressed in tissues and postnatal developmental stages examined. The presence of different N-termini in isoforms may be important for unique docking interactions with A kinase anchoring proteins.

Differentially expressed three non-coding alternate exons at 5' UTR of regulatory type I beta subunit gene of mouse.

Prkar1b gene encodes regulatory type I, beta subunit (RIß) of cAMP dependent protein kinase A in mouse. Among the various isoforms of regulatory and catalytic subunits that comprise mammalian PKA, RIß subunit is considered to be one of the important subunits for neuronal functions. This is involved in multiple forms of synaptic plasticity, and influences memory and learning by maintaining hippocampal long-term potentiation (LTP). Deficient expression of this gene has been implicated in autoimmune disease systemic lupus erythematosus (SLE). We have identified two novel non-coding exons of the Prkar1b gene (designated as exon 1A and exon 1B), which are spliced to the canonical exon 2 and constitute the 5' untranslated region giving rise to three alternative transcript isoforms. We have also confirmed the expression of the previously known first exon (designated as exon 1C) with known transcript published earlier. The transcripts containing exons 1A, 1B and 1C are differentially regulated during the development and tissue types. In silico study of more than 20 kb nucleotide sequence upstream of known translational initiation codon revealed three distinct promoter regions named as PA, PB, and PC upstream of the exon 1A, exon 1B and exon 1C respectively. PB is non-CpG related promoter but PA and PC are CpG related promoters, however all three promoters are TATA less. Further analysis showed that these promoters possess potential signature sequences for common as well as different transcription factors suggesting complex regulation of Prkar1b gene.

Alternatively spliced three novel transcripts of gria1 in the cerebellum and cortex of mouse brain.

Glutamate receptor type 1 (GluR1) subunit of α-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptors plays an important role in the expression of long-term potentiation and memory formation. GluR1 is encoded by gria1 gene containing 16 exons and 15 introns in mouse. Previous studies have reported two alternatively spliced variants of this subunit. These flip and flop variants differ enormously in their properties as well as expression. In our studies, we report the presence of three new transcripts of this gene present in the cerebellum and cortex of mouse brain produced by alternative splicing at 5' end. Four new exons are reported; N1 is located in 5' untranslated region, N2 is located in the 1st intronic region while N3 and N4 are located in the 2nd intronic region. The properties of these new exons encoding N-terminal variants are highly diverse. N1, N3 and N4 are coding while N2 is a non-coding exon and results in a truncated transcript. The existence of N2 exon containing transcript is further supported by the presence of an Expressed Sequence Tag from the database. The translated amino acid sequences of these transcripts differ in the presence of signal peptide as well as in their phosphorylation and acetylation pattern. The differences in their properties might be involved in receptor modulation.

Identification of alternatively spliced multiple transcripts of 5-hydroxytryptamine receptor in mouse.

5-Hydroxytryptamine receptors (HTRs) are coded by seventeen different genes in mouse. One of them is htr4 that codes for the HTR4 receptor, a G-protein coupled receptor containing seven transmembrane domains. In mouse, the gene is reported to contain 6 exons and 5 introns. Our present study reports the presence of four transcript variants of this gene encoding different N-termini. These transcripts are expressed in neuronal as well as non-neuronal tissues of mouse. We have identified five novel coding exons present at the 5' end of the gene which splice with the published internal exon in an alternative manner making a total of five transcripts, four new transcript variants (T1, T2l, T2s and T3) and one published earlier. All five transcripts encoding different N-termini were expressed in mouse brain. It was interesting to note the expression of only T3 transcript that was also detected in heart muscle and is the only htr4 transcript expressed in heart. For the first time a transcript of htr4 gene was detected in the heart of the mouse which might help us to make use of small laboratory animals to study HTR4 in heart. As this transcript is unique to the heart it can serve as potential therapeutic target for various cardiovascular disorders and dysregulation of heart rate, atrial contraction and atrial relaxation. These variants display heterogeneous properties in terms of the presence of signal peptide, acetylation, phosphorylation and glycosylation. Thus alternative splicing of htr4 producing heterogeneous N-termini increases the diversity of the receptor.

Alternative promoter usage and differential expression of multiple transcripts of mouse Prkar1a gene.

Prkar1a gene encodes regulatory type 1 alpha subunit (RIα) of cAMP-dependent protein kinase (PKA) in mouse. The role of this gene has been implicated in Carney complex and many cancer types that suggest its involvement in physiological processes like cell cycle regulation, growth and/or proliferation. We have identified and sequenced partial cDNA clones encoding four alternatively spliced transcripts of mouse Prkar1a gene. These transcripts have alternate 5' UTR structure which results from splicing of three exons (designated as E1a, E1b, and E1c) to canonical exon 2. The designated transcripts T1, T2, T3, and T4 contain 5' UTR exons as E1c, E1a + E1b, E1a, and E1b, respectively. The transcript T1 corresponded to earlier reported transcript in GenBank. In silico study of genomic DNA sequence revealed three distinct promoter regions namely, P1, P2, and P3 upstream of the exons E1a, E1b, and E1c, respectively. P1 is non-CpG-related promoter but P2 and P3 are CpG-related promoters; however, all three are TATA less. RT-PCR analysis demonstrated the expression of all four transcripts in late postnatal stages; however, these were differentially regulated in early postnatal stages of 0.5 day, 3 day, and 15 day mice in different tissue types. Variations in expression of Prkar1a gene transcripts suggest their regulation from multiple promoters that respond to a variety of signals arising in or out of the cell in tissue and developmental stage-specific manner.