Characterizing the Bidirectional Nature and Transcriptional Activity

Characterizing the Bidirectional Nature and Transcriptional Activity of Alternative Promoters Across Different Locations in а Gene METHODS ln past research, promoters have been found to initiate RNA transcription Ьidirectionally, meaning that nascent RNA reads exist both in the downstream sense and upstream antisense directions. ln addition to this, downstream and hybrid exons (exons that сап Ье used as first and internal exons) have been found to have an increase in usage in comparison to upstream and first exons only (exons that сап only Ье used as first exons), respectively. However, this behavior has been largely unexplored in alternative promoters, and is therefore of great interest for the advancement of the field, and the purpose of our study. • We hypothesize that in accordance with previous research, downstream and hybrid exons should display greater expression of nascent RNA than upstream and first exons only (FEO). • То test this, we aim to collect data оп the relative transcriptional activity within genes and characterize the patterns of transcription in alternative promoters for different exon classifications. • ln addition, we gain to understanding оп the frequency and scale of Ьidirectional transcription in alternative promoters. GENE COLLECTION DATA ANALYSIS ln order to compare nascent RNA reads for different exon types and genomic orders, we first produced а gene list for which to aggregate read count data. The requirements for gene and exon collection varied for exon type and genomic order respectively: 1. Up to 400 coordinate locations both downstream and upstream from the TSS were assigned for both the positive and negative strands in the gene list for nucleotide-level comparison 2. Pro-Seq data from nascent RNA read samples of Hela cells in humans were used to collect read counts for respective genes (1 > 3. Read counts across all genes for each classification (exon type ог genomic order) were then aggregated to determine the total nascent reads рег each nucleotide relative to the TSS 4. Read counts for relative exon locations were then visualized to: (1) Compare FEO vs. hybrid exons; (2) Compare genomic orders; (3) Compare downstream vs. upstream transcription • Only genes with at least 3 genomic orders (3 exons) were observed • For each gene, the first 3 exons were selected for analysis, which all were either FEO or hybrid exons first hybrid 2.5 1 direction 2.0 ё :::; о downstream upstream 1.5 ]а: • Expression in both the sense and antisense directions is greater for hybrid exons than first exons only • Results present up to 35х more nascent RNA reads in antisense direction than sense direction for hybrid exons • Маге comparative upstream expression than downstream expression for hybrid exons • Both FEO and hybrid exons have imbalanced Ьidirectionality; more upstream transcription 10.0 а: ё � 1.0 z 0.5 Existing research has displayed that promoters сап initiate transcription Ьidirectionally - in the positive and negative strand simultaneously. This is referred to as downstream 'sense' ог upstream 'antisense' transcription respectively. � un.idirectiona I prom{]ter � _·. Е;> ТТ tr#-'!'..,_ 'I_ В.: Ыdirertion.зl promoter (:"'\ о.о • --. Tf!P RNд Polll -100 -200 О 100 200 -200 Relative Location -100 О 100 200 TSP 1 RNA Polymeraзe • 1 1 :Start Codon Coding Sequence S!op Godon Promoter Figure 2: Transcriptional Start Site Genomic Order 2 Genomic Order 1 Figure 1: Bidirectional Promoter The transcriptional start site (TSS) is the location within the core promoter where transcription begins. lt provides а reference as to where exons are being transcribed from, and defines downstream versus upstream expression 2. • FEO vs. Hybrid: An exon that is only used as а first exon during transcription is а first exon only (FEO). An exon that сап Ье used as both а first and internal exon is а hybrid exon. Previous research has shown these exons to exhiblt varying transcription based оп relative location from the TSS. • Genomic Order: Genomic order describes the nth exon from upstream to downstream оп the strand being viewed. Since а hybrid exon сап always Ье an internal exon, it therefore must Ье contained in genomic order 2 or above. ... 11 200 11 DNA • Results display far greater expression in the antisense direction for both genomic orders 1 and 2 Genomic Order 3 dowпstream upstream ё 8 ]а: .,z а: 10 о -50 О 50 100-100 -50 О 50 Relative Location 100-100 -50 О Figure 7: Nascent RNA Reads for Genomic Orders 11 -100 Relalive Locatlon -50 • to Sense Direction for Exon Туре 20 -100 .,50 Figure б: Antisense RNA Reads Proportional Figure 5: Nascent RNA Reads for Exon Туре R.Nд11 Pol 1 Site (ТSS) О.О -t::;, direction Tmnscriplion SlaJ! '' 50 100 • lncrease in upstream expression from genomic order 1 to 2, and decrease from genomic order 2 to 3 • No visiЫe spike in upstream • • express1on 1n genomic order 3, up to 200х more а: antisense reads < z а: for genomic order j 2 • Greatest activity across all genomic orders near 25th upstream nucleotide from TSS Gooom� Order �э�� Oroer 1 (Э�� Qfder 2 - Gel)QПiC Oцier 3 150 100 50 • -100 -15 -50 Aelati� L.ooe1tion -2� о Figure 8: Antisense RNA Reads Proportional to Sense Direction for Genomic Orders pro(ein � Figure 3: FEO vs. Hybrid Exons . ,�••--;........-.;;C=.;;;;81-a;;;O=------j�,•�- ----- ,....,3 1-1 ,__ ....,,) ._. -1 ,_, - Gene (DNA) ......,,,__ Figure4: Exon Genomic Order ln summary, we find that for all genomic order exons in а gene, nascent RNA transcription was more common in the upstream antisense direction than the downstream sense direction. Upstream gene expression increased between the first and second genomic order exons along а gene, but soon dropped in the third genomic order exon. Finally, transcription in both the sense and antisense direction occurred at higher rates in hybrid exons as compared to first exons only (FEO), concurring with our hypothesis and previous research in the field. This study entails further investigation into why transcriptional disparities exist among alternative promoters. The observations provided allow for а greater understanding of how alternative promoters exhiblt Ьidirectional transcription, and take а step in that direction.