Origins and Impacts of New Mammalian Exons

Mammalian genes are composed of exons, but the evolutionary origins and functions of new internal exons are poorly understood. Here, we analyzed patterns of exon gain using deep cDNA sequencing data from five mammals and one bird, identifying thousands of species-and lineage-specific exons. Most new exons derived from unique rather than repetitive intronic sequence. Unlike exons conserved across mammals, species-specific internal exons were mostly located in 5' UTRs and alternatively spliced. They were associated with upstream intronic deletions, increased nucleosome occupancy, and RNA polymerase II pausing. Genes containing new internal exons had increased gene expression, but only in tissues in which the exon was included. Increased expression correlated with the level of exon inclusion, promoter proximity, and signatures of cotranscriptional splicing. Altogether, these findings suggest that increased splicing at the 5' ends of genes enhances expression and that changes in 5' end splicing alter gene expression between tissues and between species.Peer reviewe

Alternative Splicing of RNA Triplets Is Often Regulated and Accelerates Proteome Evolution

Thousands of human genes contain introns ending in NAGNAG (N any nucleotide), where both NAGs can function as 3′ splice sites, yielding isoforms that differ by inclusion/exclusion of three bases. However, few models exist for how such splicing might be regulated, and some studies have concluded that NAGNAG splicing is purely stochastic and nonfunctional. Here, we used deep RNA-Seq data from 16 human and eight mouse tissues to analyze the regulation and evolution of NAGNAG splicing. Using both biological and technical replicates to estimate false discovery rates, we estimate that at least 25% of alternatively spliced NAGNAGs undergo tissue-specific regulation in mammals, and alternative splicing of strongly tissue-specific NAGNAGs was 10 times as likely to be conserved between species as was splicing of non-tissue-specific events, implying selective maintenance. Preferential use of the distal NAG was associated with distinct sequence features, including a more distal location of the branch point and presence of a pyrimidine immediately before the first NAG, and alteration of these features in a splicing reporter shifted splicing away from the distal site. Strikingly, alignments of orthologous exons revealed a ~15-fold increase in the frequency of three base pair gaps at 3′ splice sites relative to nearby exon positions in both mammals and in Drosophila. Alternative splicing of NAGNAGs in human was associated with dramatically increased frequency of exon length changes at orthologous exon boundaries in rodents, and a model involving point mutations that create, destroy, or alter NAGNAGs can explain both the increased frequency and biased codon composition of gained/lost sequence observed at the beginnings of exons. This study shows that NAGNAG alternative splicing generates widespread differences between the proteomes of mammalian tissues, and suggests that the evolutionary trajectories of mammalian proteins are strongly biased by the locations and phases of the introns that interrupt coding sequences.Damon Runyon Cancer Research Foundation (DRG 2032-09)National Science Foundation (U.S.). (no. 0821391)United States. National Institutes of Healt

Research and Design of a Routing Protocol in Large-Scale Wireless Sensor Networks

无线传感器网络，作为全球未来十大技术之一，集成了传感器技术、嵌入式计算技术、分布式信息处理和自组织网技术，可实时感知、采集、处理、传输网络分布区域内的各种信息数据，在军事国防、生物医疗、环境监测、抢险救灾、防恐反恐、危险区域远程控制等领域具有十分广阔的应用前景。 本文研究分析了无线传感器网络的已有路由协议，并针对大规模的无线传感器网络设计了一种树状路由协议，它根据节点地址信息来形成路由，从而简化了复杂繁冗的路由表查找和维护，节省了不必要的开销，提高了路由效率，实现了快速有效的数据传输。 为支持此路由协议本文提出了一种自适应动态地址分配算——ADAR（AdaptiveDynamicAddre...As one of the ten high technologies in the future, wireless sensor network, which is the integration of micro-sensors, embedded computing, modern network and Ad Hoc technologies, can apperceive, collect, process and transmit various information data within the region. It can be used in military defense, biomedical, environmental monitoring, disaster relief, counter-terrorism, remote control of haz...学位：工学硕士院系专业：信息科学与技术学院通信工程系_通信与信息系统学号：2332007115216

Specific intronic sequence features regulate NAGNAG splicing.

<p>(A) Illustration of NAGNAG minigene constructs, designed to test the roles of the branch point to 3′ splice site distance and of the −4 base in NAGNAG splicing. A short segment of intronic sequence spanning the branch point to the 3′ splice site of the PTBP2 NAGNAG was cloned upstream of the IGF2BP1 exon. To confirm the importance of a pyrimidine at the −4 position for distal NAG use, the effects of all four nucleotides at the −4 position were tested. The branch point to 3′ splice site distance was varied by introducing nucleotides (underlined in orange) in constructs containing the PTBP2 branch point sequence, or by removing nucleotides (indicated by green dots) in constructs containing the IGF2BP1 branch point sequence. Locations of RT-PCR primers are indicated by arrows. (B) Proximal isoform expression increased dramatically after the introduction of a purine at the −4 position. Splicing was monitored after minigene transfection into HEK293T cells by RT-PCR. Mean and standard deviation of at least three independent transfections are shown. A representative gel is shown below (top and bottom bands represent proximal and distal isoforms, respectively). (C) As in (B), but varying the branch point to 3′ splice site distance in the context of the native nPTB branch point sequence. The distance was increased by insertion of four or seven nucleotides of sequence of varying purine/pyrimidine composition as shown in (A). (D) As in (C), but decreasing the branch point to 3′ splice site distance in the context of the exogenous IGF2BP1 BPS by deletion of three or six bases as shown in (A).</p

Alternative splicing of tissue-specific NAGNAGs is highly conserved.

<p>(A) Short reads were aligned to the intron-proximal and intron-distal splice junctions of NAGNAG splicing events in order to estimate isoform ratios. (B) Estimated proximal isoform usage (ψ) for a NAGNAG which inserts/deletes a predicted phosphorylation site in far upstream element binding protein 1 (FUBP1). Phosphorylation site and corresponding kinase were predicted by Scansite (Scansite <i>z</i>-score −3.024) <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001229#pbio.1001229-Obenauer1" target="_blank">[55]</a>. Error bars indicate the 95% binomial confidence interval. (C) Number of reading frame-preserving alternative splicing events in protein-coding regions, with both isoforms expressed at ≥5% in at least one tissue (see also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001229#pbio.1001229.s012" target="_blank">Table S1</a>). (D) A NAGNAG which inserts/deletes an arginine in RNA recognition motif 4 (RRM4) of the splicing factor PTBP2 is deeply conserved. Alignment of orthologous 3′ splice site sequences shown below the NMR structure (PDB accession 2ADC, displayed with PyMOL) of the highly homologous PTBP1 protein (green) complexed with RNA (red) <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001229#pbio.1001229-Oberstrass1" target="_blank">[33]</a>. Boxed is K489 of PTBP1, which is homologous to the arginine shown in PTBP2, and hydrogen bonds to the RNA backbone (dotted yellow line). Putative branch point based on location of the first upstream AG, the sequence motif identified in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001229#pbio.1001229-Reed1" target="_blank">[56]</a>, and the pattern of sequence conservation. (E) Conservation of alternative splicing between orthologous human and mouse NAGNAGs increases with tissue specificity. NAGNAGs that were alternatively spliced in human (left) and mouse (right) were grouped by switch score—defined as the maximum ψ difference between tissues—as indicated by colors, and the fraction of orthologs which were alternatively spliced in the other species is shown. Error bars indicate 95% binomial confidence intervals.</p

NAGNAGs are associated with accelerated protein evolution at exon-exon boundaries.

<p>(A) Alignment of portions of exons 10 and 11 of TRIM28 gene from three mammals, illustrating a shift in the upstream boundary of exon 11 between human and rodents. Exonic sequence shown in capitals; intronic sequence in lower case. (B) Gain/loss of exonic sequence between human and mouse occurs preferentially at 3′ splice sites (<i>p</i><10⁻⁶, permutation test). The fraction of aligned orthologous human and mouse exons with gaps at each position is shown; the background level (mean fraction across the indicated region excluding the 3′ splice site) is shown by the dotted yellow line; the right-hand axis shows enrichment relative to this background. (C) As in (B), but restricted to gaps of length three bases. Preferential occurrence at 3′ splice sites was highly significant (<i>p</i><10⁻⁶, permutation test). (D) Similar to (C), but based on alignments of orthologous <i>D. melanogaster</i> and <i>D. yakuba</i> exons. Preferential occurrence at 3′ splice sites was highly significant (<i>p</i><10⁻⁶, permutation test). (E) Similar to (C), but based on alignments of orthologous <i>C. elegans</i> and <i>C. briggsae</i> exons. Preferential occurrence at 3′ splice sites was highly significant (<i>p</i><10⁻⁶, permutation test). (F) Residual NAG motif at exons whose boundaries changed in the rodent lineage. Orthologous mouse and rat exons were classified as unchanged (top), expanded by three bases (middle), or contracted by three bases(bottom) based on comparison to an outgroup (human, cow, chicken, or <i>Xenopus laevis</i>), aligned to the inferred location of the ancestral 3′ splice site (dotted line). Information content of each position is shown relative to a uniform background composition. (G) Exons whose 3′ splice site boundaries differ by three bases between rat and mouse are 7.5 times as likely to have a NAGNAG in the human ortholog as exons whose boundaries did not change (<i>p</i>-value for difference<10⁻²⁴ by Fisher's exact test). Error bars indicate the 95% binomial confidence interval. (H) Rodent exons orthologous to alternatively spliced human NAGNAG exons (left) are much more likely to exhibit exon boundary changes of three base pairs than those orthologous to constitutively spliced human NAGNAGs (right) (<i>p</i>-value for difference<10⁻¹⁰ by Fisher's exact test). Blue and gray bars in (H) represent subsets of blue and gray bars in (G), respectively. Error bars indicate the 95% binomial confidence interval. (I) Frequency of encoded amino acids that occur opposite gaps at the 3′ splice site in alignments of human and mouse exons is plotted above, overall (pink) and separately by the phase of the upstream intron (i.e., the number of bases, if any, in the last incomplete codon of the upstream exon); amino acid frequency at background positions (4 codons downstream of the 3′ splice site) is shown below. The Shannon entropy (a measure of randomness) of each amino acid frequency distribution is also shown.</p

Increased sequence conservation upstream of tissue- and developmentally-regulated NAGNAGs.

<p>(A) NAGNAG ψ estimates are highly consistent in brain RNA-Seq data from the mouse strains DBA/2J and C57BL/6J. Only NAGNAGs with both isoforms expressed at ≥5% in either strain are shown. The 75^th percentile of the deviation from the line <i>y = x</i> is shown in gray. (B) NAGNAG ψ estimates are quantitatively conserved between human and mouse brain. Only NAGNAGs with both isoforms expressed at ≥5% in either species and satisfying |proximal 3′ splice site score – distal 3′ splice site score|≤0.5 bits are plotted (splice sites scored by MaxEnt model <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001229#pbio.1001229-Yeo1" target="_blank">[36]</a>). Deviation from <i>y = x</i> shown as in (A). (C) Sequence conservation of human NAGNAGs, where all NAGNAGs are aligned by their 3′ splice site junctions and grouped by switch score. Mean (solid line) and standard error of the mean (shaded area about solid line) of phastCons score <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001229#pbio.1001229-Siepel1" target="_blank">[50]</a> shown by position (averaged over a 2 nt sliding window) for each switch score category. Analysis restricted to human NAGNAGs for which the two AGs were conserved at the sequence level in mouse. (D) As in (C), but grouped by switch score in mouse and restricted to mouse NAGNAGs for which the two AGs were conserved at the sequence level in human. (E) As in (D), but for NAGNAGs in <i>Drosophila melanogaster</i>, with switch score defined across developmental stages rather than between tissues. Analysis restricted to <i>D. melanogaster</i> NAGNAGs for which the two AGs were conserved at the sequence level in <i>D. yakuba</i>.</p

RNA Sequence Context Effects Measured In&nbsp;Vitro Predict In&nbsp;Vivo Protein Binding and Regulation

Many RNA binding proteins (RBPs) bind specific RNA sequence motifs, but only a small fraction (∼15%-40%) of RBP motif occurrences are occupied in&nbsp;vivo. To determine which contextual features discriminate between bound and unbound motifs, we performed an in&nbsp;vitro binding assay using 12,000 mouse RNA sequences with the RBPs MBNL1 and RBFOX2. Surprisingly, the strength of binding to motif occurrences in&nbsp;vitro was significantly correlated with in&nbsp;vivo binding, developmental regulation, and evolutionary age of alternative splicing. Multiple lines of evidence indicate that the primary context effect that affects binding in&nbsp;vitro and in&nbsp;vivo is RNA secondary structure. Large-scale combinatorial mutagenesis of unfavorable sequence contexts revealed a consistent pattern whereby mutations that increased motif accessibility improved protein binding and regulatory activity. Our results indicate widespread inhibition of motif binding by local RNA secondary structure and suggest that mutations that alter sequence context commonly affect RBP binding and regulation

Germline quality control: eEF2K stands guard to eliminate defective oocytes

The control of germline quality is critical to reproductive success and survival of a species; however, the mechanisms underlying this process remain unknown. Here, we demonstrate that elongation factor 2 kinase (eEF2K), an evolutionarily conserved regulator of protein synthesis, functions to maintain germline quality and eliminate defective oocytes. We show that disruption of eEF2K in mice reduces ovarian apoptosis and results in the accumulation of aberrant follicles and defective oocytes at advanced reproductive age. Furthermore, the loss of eEF2K in Caenorhabditis elegans results in a reduction of germ cell death and significant decline in oocyte quality and embryonic viability. Examination of the mechanisms by which eEF2K regulates apoptosis shows that eEF2K senses oxidative stress and quickly downregulates short-lived antiapoptotic proteins, XIAP and c-FLIPL by inhibiting global protein synthesis. These results suggest that eEF2K-mediated inhibition of protein synthesis renders cells susceptible to apoptosis and functions to eliminate suboptimal germ cells