Laboratory Research Interests

RNA shows greater-than-expected roles in gene regulation by expanding the eukaryotic proteome through alternative splicing and by diversifying genetic outputs through the expression of long non-coding transcripts that impact gene expression at a variety of levels. The long-term goal of my laboratory is to understand RNA complexity and regulatory networks in higher eukaryotic cells at the whole transcriptome level by taking advantage of state-of-the-art deep sequencing approaches and new bioinformatic analyses.

Specific aim 1: Decipher alternative splicing networks.

The vast majority of eukaryotic genes contain introns that are removed by the process of pre-mRNA splicing. Indeed, most mRNA precursors in higher eukaryotes contain multiple exons and introns, and in such cases the exons can be joined together in different patterns in a process called alternative splicing to generate mature mRNAs. Alternative splicing is an important mechanism of gene regulation in higher eukaryotes, and contributes significantly to proteomic complexity and thereby functional diversity. We have combined RNAi and mRNA-seq to identify exons that are regulated by Pasilla (PS), the D. melanogaster ortholog of mammalian splicing factors NOVA1 and NOVA2. Very interestingly, the RNA regulatory map of PS and NOVA1/2 is highly conserved between insects and mammals suggesting that the regulatory modules controlled by these proteins are highly evolvable. In my laboratory, we will continue studying the global impact of alternative splicing changes regulated by PS and dozens of other specific protein regulators using high throughput sequencing methods combined with genetic, biochemical, and bioinformatic strategies.

Specific aim 2: Discover new modes of gene regulation and new noncoding RNA species.

Despite the fact that over 80% of the human genome is transcribed, the great majority (more than 98%) of the transcriptional output represents non-protein coding RNAs (ncRNAs). In recent years, long ncRNAs (lncRNAs, >200 nt) have emerged as a new class of RNAs with various functions and specific cellular locations. We have recently explored the repertoire of non-polyadenylated RNAs genome-wide from both human embryonic stem cells (hESCs) and carcinoma cell lines by a newly developed RNAseq method, and have identified a large number of novel non-polyadenylated lncRNAs that are derived from intronic regions. Many of these are expressed from disease-related genes or disease-related genomic regions. These recent research accomplishments have opened a number of new and distinct directions in the lncRNA field. Importantly, some of these new lncRNAs appear to be processed in a manner not described before in the literature. In my laboratory, we will investigate the processing mechanism of non-polyadenylated RNAs by computing consensus motifs withinin and near their sequences. We will also explore their possible functions by examining their correlation with small RNA precursors, such as microRNAs. In addition, we are also interested in their evolutionary origins. Finally, we will also apply our methods to the analysis of lncRNAs during hESC differentiation to different lineages, which will add more depth to our current understanding of RNA complexity related to pluripotency, self-renewal and differentiation.

Our studies to identify alternatively spliced exons and non-polyadenylated lncRNAs will have a profound impact on the understanding of RNA complexity and its functions in eukaryotes.

405 PS-regulated pre-mRNA processing events. (Black boxes) Constitutive regions; (white boxes) alternative regions. (Red lines) Splice junctions for the inclusion isoform; (blue lines) junctions for the exclusion isoform. (Red bars) Exonic reads that support the inclusion isoforms; (blue bars) exonic reads that support the exclusion isoforms; (red bars with a black line) reads that support the inclusion isoform, but have a shared portion with the exclusion isoform. Thinner portions of the boxes in alternative first exons and alternative last exons correspond to UTRs. Brooks et al. Genome Res., 2011, 21:193-202

Discovery of small non-coding RNAs. a, Poly(A)1 (yellow) and total RNA (blue) data from 10–12-h embryos are shown for the gp210 gene which hosts a representative new snoRNA. The maximalnumber of reads in the poly(A)1 and total RNA-Seq data are shown on the left and right of the track, respectively. b, The predicted RNA secondary structure of snoRNAgp210 is characteristic of a H/ACA-box snoRNA. Nucleotides that are 100% conserved in sequence or base-pairing are indicated in green and blue, respectively. c, Embryonic expression of the new small RNAs. The scale bar indicates FPKM Z-scores. unsRNA, unclassified small RNA. Graveley et al. Nature, 2011, 471:473-479

Classification of poly(A)- transcripts. (a) Classification of poly(A)- transcripts: EIs, excised introns; ZNF, zinc finger factor protein family. See text for details. (b) Overlapping analysis of the expression of poly(A)- transcripts in H9 and HeLa cells. (c) Example of a poly(A)- non-histone mRNA, znf460 and sesn3. The relative signals from either poly(A)+ or poly(A)- RNA preparations were normalized to those in the total RNA preparation in each cell line. Note that the signals from the poly(A)- samples are significantly enriched. Black arrows show the extended unannotated 3' UTR region of znf460. (d) An example of the excised 16th intron of the mRNA azi1. The blue box reveals the information in detail from this region. Note that the excised intron is abundant and can be detected only in the poly(A)- samples. (e) Examples of excised introns from different mRNAs, and the position of the excised intron in each mRNA is indicated. Yang et al. Genome Biol., 2011, 12(2):R16