AS represents a mechanism for gene regulation and expansion of the proteome. These factors generally regulate AS by recognizing cis -acting sequences in exons or introns and by promoting or suppressing the assembly of the spliceosome at adjacent splice sites. Studies in yeast and metazoan systems have indicated that the levels of some of these core splicing components can affect splice site choice.
Microarray profiling revealed transcript-specific effects on splicing in yeast strains harboring mutations in or deletions of core splicing components Clark et al.
Knockdown of several core splicing factors in Drosophila cells resulted in transcript-specific effects on AS reporters Park et al. Tiling microarray profiling analysis of fission yeast RNA also revealed transcript-specific splicing defects of a temperature degron allele of SMN, and that some of the defects could be alleviated by strengthening the pyrimidine tract upstream of the branchpoint Campion et al.
However, the features that underlie the differential sensitivity of introns or alternative exons to particular defects in the core splicing machinery are not well understood. Such autoregulation and cross-regulation are important for the establishment and maintenance of AS factor levels across different tissues and developmental stages. Autoregulation and cross-regulation of AS factors can produce protein isoforms with different functional properties Dredge et al. Consistent with their functional importance, regulated AS events involved in AS-NMD of splicing factors often lie within highly conserved or ultraconserved sequence regions Lareau et al.
Using AS microarray profiling following knockdown of NMD factors, we previously identified highly conserved, PTC-introducing alternative exons in genes encoding multiple core splicing factors Saltzman et al.
These results suggested that AS-NMD plays a role in the homeostatic regulation not only of AS regulatory factors, but of components of the basal splicing machinery as well. Moreover, the results further suggested that core spliceosomal components may regulate specific AS events in addition to their well-studied critical roles in constitutive splicing.
This alternative exon and its conserved flanking intron regions boxed in blue were cloned into a minigene miniSmB in which they are flanked by heterologous intron and exon sequences.
See also Supplemental Figures 2 and 3. Cells were then cotransfected with miniSmB and either an empty vector or a vector encoding a 3xFlag-tagged cDNA, as indicated above the gels. Top panel To assay alternative exon inclusion in miniSmB, RT—PCR assays were performed using primers specific for the flanking constitutive exons of the minigene hatched boxes.
Our results thus reveal an important role for the core spliceosomal machinery in establishing the inclusion levels of a specific subset of alternative exons, and further suggest that changes in the levels of these alternative exons control the expression of other RNA processing factors.
This exon, together with its highly conserved flanking intronic sequences, was cloned into a minigene reporter plasmid containing upstream and downstream heterologous intron and constitutive exon sequences Fig. Our results further suggest that components of the core spliceosomal machinery can function in the regulation of AS, in addition to their well-established roles in constitutive splicing.
Taken together with the results described above, these observations suggest that SmB and SmD1 affect AS in a similar but nonredundant manner. Cells were then cotransfected with miniSmB and with either an empty vector or a vector encoding the 3xFlag-tagged cDNA indicated above the gel. The quantifications and bar graph of percent inclusion levels are as in Figure 1. The similar effects observed for both knockdowns are consistent with a shared role for Sm proteins in the inclusion of alternative exons through modulating the overall levels of one or more snRNPs.
Recapitulation in this reporter Fig. Linker-scanning mutagenesis was performed, in which successive base segments of the alternative exon and its upstream and downstream flanking introns were deleted or substituted with a linker sequence. This strategy identified sequences in the exon and introns acting as splicing enhancers or silencers.
These elements are concentrated near splice sites Supplemental Fig. These results suggest that these sequence motifs either act through different trans -acting factors, or are involved in regulation in a manner that is redundant with other sequence motifs. Serial dilutions of protein extract indicate that the blots are semiquantitative.
Counts of reads mapping to included versus skipped exon junctions were used to calculate the percent inclusion level of these alternative exons. In contrast, knockdown of SRSF1 resulted in 7. Only 1. A Scatter plot showing agreement between percent inclusion of 27 alternative exons in the three knockdowns as measured by RT—PCR versus RNA-seq left , and between differences in inclusion levels knockdown relative to control NT for the same 27 alternative exons right.
The number following the period designates the AS event ID. Names with an asterisk PTC upon skipping. Consistent with previous results Stamm et al. Similar results were also obtained using Pathway Commons annotations Cerami et al. These results therefore support the conclusion that an important role for alternative exons affected by changes in the level of the core spliceosomal snRNP machinery is to coordinately control the expression of many RNA processing factors and other regulators of RNA.
Each node represents the set of genes annotated with the indicated GO term. Node size is proportional to the number of genes annotated by the term indicated by the node label , and edge thickness is proportional to the number of genes in common between the sets. B Model summarizing our data. In this study, we identify a network of alternative exons in RNA processing factor genes that is controlled by the levels of the core spliceosomal machinery.
We provide insight into how these exons are regulated, and their roles in both feedback and coordinated control of gene expression summarized in Fig. Previous studies have shown that mutation, deletion, or knockdown of core spliceosomal and spliceosome assembly factors can result in altered splicing patterns in yeast Clark et al.
Thus, as is well established for splicing regulatory factors, it is emerging that the relative concentration or activity of general splicing factors can affect splice site selection.
Consistent with an important physiological role for the core spliceosomal machinery in the regulation of AS, components of snRNPs are differentially expressed in mammalian cells and tissues Grosso et al. Evidence for critical physiological regulatory roles for core spliceosomal components and assembly factors have also emerged from the study of certain human diseases.
U5 tri-snRNP particle are associated with retinitis pigmentosa for review, see Mordes et al. Although the specific mechanisms and transcript targets that are responsible for these diseases are largely unknown, these studies point to the importance of maintaining appropriate expression of the core splicing machinery. These findings may relate to previous observations revealing that the Sm complex contributes to the stability of the U1 snRNA:pre-mRNA interaction in yeast Zhang et al.
Furthermore, our data support evidence that altering the kinetics of spliceosomal rearrangements can affect splice site selection Query and Konarska ; Yu et al. Such kinetic competition between splice sites may provide a basis for the changes in the alternative exon inclusion levels that we observe in this study, where specific splice sites are no longer efficiently recognized when core splicing components, normally present at saturating levels, may become rate-limiting Smith et al.
These results extend previous observations of feedback and cross-regulation among specific splicing factors via AS-NMD.
For example, in addition to several reported examples of feedback regulation of splicing components and other RNA-binding proteins for review, see Lareau et al. The expression of SmN is often disrupted in Prader-Willi syndrome PWS , a disorder with a range of symptoms, including cognitive impairment for review, see Cassidy and Driscoll However, in brain tissue from PWS individuals or mouse models lacking SmN expression, SmB expression is up-regulated through a previously unknown mechanism Yang et al.
Our results show that these highly similar proteins have overlapping functions, and therefore are capable of cross-regulation via AS-NMD in vivo. It is also interesting to consider that cross-regulation of these paralogs via AS-NMD may reduce the phenotypic severity of loss of SmN expression. In summary, we uncovered a large set of alternative exons that are controlled by levels of the core spliceosomal machinery.
A subset of the affected exons likely plays a critical role in maintaining balanced levels of splicing and other RNA-associated factors. These results thus provide new insight into regulated exon networks as well as the functions of core spliceosomal components. For knockdowns, cells were transfected with siRNAs On-TargetPlus-modified, Dharmacon at a final concentration of nM using Dharmafect1 Dharmacon according to the manufacturer's instructions.
Plasmids were transfected using Lipofectamine Invitrogen according to the manufacturer's instructions. Cells were then harvested 2 d after the plasmid transfection. Treatment with cycloheximide and mRNA half-life experiments are described in the legends for Supplemental Figures 2 and 3. Primer sequences for AS events are available on request. All constructs were verified by sequencing. The mRNA-seq reads were mapped to exon—exon junction sequences in this database of cassette AS events as described Pan et al.
In parallel, sequencing reads were also aligned to RefSeq transcripts, and transcript levels were estimated using the reads per kilobase of exon per million mapped reads RPKM calculation Mortazavi et al. Reads were aligned to the exon—exon junctions and filtered as described above for the AS events. Enrichment of GO Ashburner et al. A minimum of 10 genes per category were specified and enrichment P -values were calculated using the hypergeometric test, and were adjusted for multiple testing using the false discovery rate FDR Benjamini and Hochberg Three nodes for GO terms with an identical set of 14 genes were collapsed into the single gene node shown Fig.
Sample sizes are given in the text. To compare the median splice site strengths, the lengths of profiled exons, and the changes in transcript levels between subsets of AS events, the nonparametric Wilcoxon rank sum test was used.
Gene Expression. Protein Structure. Protein Function. Analyzing Regulatory Networks in Bacteria. Aging and Cell Division. Calreticulin: a Multifaceted Protein. Enzyme Catalysis: The Serine Proteases. Germ Cells and Epigenetics. MicroRNAs in Arabidopsis. Protein Misfolding and Degenerative Diseases. Systems Biology of the Cell.
Joint Science Dept. Citation: Edwalds-Gilbert, G. Nature Education 3 9 How can only 25,, protein-coding genes in humans produce the massive variety of proteins, cells, and tissues that exist in our bodies? The answer: alternative splicing. Aa Aa Aa. Figure 1: A schematic representation of alternative splicing. The figure illustrates different types of alternative splicing: exon inclusion or skipping, alternative splice-site selection, mutually exclusive exons, and intron retention.
Figure 2. Alternative splicing of pre-mRNAs contributes to the diversity of proteins in eukaryotes. Cells respond to signals from the environment through changes in gene expression and protein activity, both of which play important roles in the regulation of alternative splicing. Understanding the interplay between cell signaling, cell cycle, and alternative splicing is an active area of research that requires a variety of experimental approaches. References and Recommended Reading Barash, Y.
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Change LearnCast Settings. Loci regulating isoform expression or splice junction usage were distinct from each another Figure 6.
Note that a given splice junction may be associated with more than one AS event. Figure 6. In this study we investigated the genetic regulation of alternative splicing and its functional consequences in a sample of genetically unrelated individuals of the non-domesticated tree species P.
We found that AS is self-regulating via the gain and loss of protein domains and via the gain and loss of miRNA binding sites in RNA recognition motif containing genes. We also found that contrary to the genetic regulation of transcript expression, the genetic regulation of splice junction usage is trans rather than cis regulated.
This study increases the understanding of population scale alternative splicing in the Populus genus previously found by Bao et al. Additionally, it provides perspective into the heritability of alternative splicing by the proportion of isoform expression variance explained by the genetic variance in the samples under investigation. To our knowledge, this is the first study to investigate the genetic regulation of alternative splicing and provide a comprehensive overview of its downstream consequences in a forest tree species.
By leveraging multiple assembly programs, followed by stringent splice junction read support filtering, we generated a robust transcriptome representing all observed isoforms expressed in differentiating xylem, and calculated the extent of their genetic regulation. The downstream consequences of these splicing events were also investigated using the predicted in silico translated peptide sequences generated. Finally, loci that contribute to the expression of transcript isoforms and splice junction usage were detected.
An initial analysis of transcriptome showed that 1, of the expressed genes identified represent potentially novel, unannotated open reads frames. The discovery of new transcriptional units not present in a reference genome is common when performing transcriptome assemblies Schliebner et al.
Moreover, many of these new transcriptional units are potentially tissue-specific and expressed only in xylem. While AS has been extensively characterized across different species and tissues Chamala et al. By characterizing isoform expression in a P. While large, this proportion is contrastingly smaller relative to the gene-level heritability i. Lower heritability at the isoform level suggests that there may be more limited genetic regulation i.
We discarded this hypothesis by quantifying the number of isoforms that are heritable for each individual gene that contained multiple isoforms and observed that in most cases two or more isoforms have a heritability significantly higher than 0. We also measured the relationship between expression of isoforms and their heritability, considering that weakly transcribed isoforms may be unreliably quantified therefore resulting in lower heritabilities or the consequence of regulation driven by non-genetic factors.
However, we detected very limited correlation between the level of expression of isoforms and their heritability — that is, more highly expressed isoforms are not necessarily the most heritable. While the heritability provides an estimate of the extent of variation attributable to a genetic component, the genotypic data available for this population created the opportunity to also identify iso-eQTL and sQTL.
Based on this analysis, the majority of iso-eQTL were designated as cis based on their proximity to the transcript they were regulating while the majority of sQTL were trans. In eQTL studies it is often found that a large fraction of genes are regulated in cis Ranjan et al. In fact, only 95 genes had their different isoforms distinctly regulated in both cis and trans. The majority of sQTL however, were trans. Previous sQTL studies in humans focused their analysis only to variants that were proximal, or in cis , to AS genes Li et al.
Our findings indicate that variants regulating splice junction usage that are distant from the AS genes should be considered when investigating sQTL. Isoform expression has been often shown to be tissue specific — that is, a single isoform of an AS gene is predominately expressed in a given tissue Wang et al. Tissue specific expression of alternatively spliced isoforms plays roles in plant growth, development, and the establishment of tissue identity Staiger and Brown, While this study evaluated isoform expression exclusively in differentiating xylem, the analysis of a large population allowed us to assess if specific isoforms are predominant or if multiple, alternative isoforms may be the most expressed in different individuals of the population.
Therefore, most genes have a single isoform that constitutes the majority of transcript expression. This result was also similar to the analysis by Bao et al. Further work that investigates isoform frequencies using RNA-seq libraries from multiple plant tissues in a diverse population will determine if other isoforms are the most highly expressed in different tissues. Alternative splicing increases the complexity of the proteome without changes to the genome by producing multiple mRNA species from a single gene.
Changing the sequence of an mRNA can change the sequence of the peptide translated from it. Previous AS studies in plants have uncovered AS events affecting specific protein domains Thatcher et al.
However, to the best of our knowledge, a comprehensive overview of protein domain architecture modified by AS events in plants has not been reported. Enrichment analysis for the genes with modified domain architecture revealed that only genes involved in RNA binding GO term: nucleic acid binding and PFAM: RNA recognition motif were enriched among the set of genes affected by this process. The gain or loss of miRNA binding sites as a result of AS between tissues and developmental stages in maize was reported previously Thatcher et al.
Our analysis revealed that the most highly expressed isoform of a gene more often lacked a miRNA binding site that lower expressed isoforms of the gene contained. This finding may be the result of post-transcriptional down-regulation of minor isoforms of genes by the miRNAs targeting minor isoforms.
PCK was found to be upregulated in Populus hybrids exposed to high levels of nitrogen whose xylem architecture was remodeled as a result of this treatment Plavcova et al.
These four genes were highly expressed in the population median FPKM: Further investigation of these transcripts and their miRNA binding sites may reveal effects on xylem architecture and nitrogen use. The analyses in this study provides a description of alternative splicing in a large sample of unrelated individuals of P.
A previous study in Populus investigated AS in a population of 20 genotypes Bao et al. Here we expand this by profiling AS at the population transcriptome level, identifying putative genetic regulators of AS, and identifying the AS events that affect the gain or loss of protein domains and miRNA binding sites.
The genes with genetically regulated AS and protein domain changes resulting from AS may be incorporated into genomic selection models to improve their robustness via serving as markers for selection.
These genes may also be further analyzed for their potential role on phenotypic traits. JN performed isoform assemblies and related bioinformatics analysis. MR assisted in heritability estimations. NSF award number to WB. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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