Derek de Rie and Imad Abuessaisa Presented by: Cassandra Derrick An integrated expression atlas of miRNAs and their promoters in human and mouse Derek de Rie and Imad Abuessaisa Presented by: Cassandra Derrick

Goal of the paper Create an expression atlas of promoters and miRNAs from mice and human samples Used sRNA sequencing libraries and CAGE data Focused on human primary cells sRNA= small RNA

Background miRNA = microRNA Short (21 – 23 nt) Noncoding Bind 3’ UTR of complementary sequences – reduce translation/repression of mRNA Involved in cellular regulation FANTOM5 project – Functional Annotation of Mammalian Genome 5th generation Worldwide project to characterize mammalian genomes Order: miRNA gene -> pri-miRNA (primary) -> pre-miRNA (nucleus) -> pre-miRNA (cytoplasm) -> mature miRNA (uses Drosha and DICER)

Methods sRNA libraries – Illumina TruSeq Sample Prep protocol Same RNA samples were used for CAGE libraries Drosha CAGE peak analysis CAGE = Cap Analysis Expression Data Calculated tags at each genomic position Defined 3’ end of pre-miRNA: 3’ nt of mature miRNA on 3’ arm of the pre- miRNA Miru28 visualization – cluster and visualization of miRNA expression Validation of miRNA expression – qPCR Validation of miRNA promoters – RAMPAGE and RAGE Normalized miRNA expression data ENCODE RAMPAGE – sequencing data PAGE – PCR protocol – rapid amplification of gene ends Matching CAGE library to the sRNA

sRNA and Criteria iPS = induced pluripotent cells ES = embryonic cells Focused on primary cells (table 1) First criteria needed to be met for 2-4 to be evaluated Importance – position and mature miRNA on both strands of pre-miRNA Criteria – defined based on expression and structural characteristics

Criteria satisfied by pre-miRNA in miRBase Robust set – 4/5 criteria met Permissive set – remaining pre-miRNA Robust set – 795 (human) and 502 (mouse) Permissive set – 1,076 (human) and 684 (mouse) 571 human pre-miRNAs satisfied all 5 criteria Divided into two sets based on criteria met Robust set – 4/5 met Permissive set – remaining pre-miRNA Half of robust set had significant Drosha CAGE peaks Mirtrons = miRNA located in introns

Drosha CAGE peak analysis Drosha CAGE peak – 3’ end of the pre-miRNA CAGE peak marks Drosha cleavage site Zebra fish – Drosha cleavage site at the end of 3’ of pre-miRNA, characterized by distinctive CAGE peak Difference between ENCODE CAGE and FANTOM5 CAGE data – due to difference in sequencing techniques Genomic locus – mir-223 in human CAGE tags Drosha CAGE peak – 3’ end of the pre-miRNA Peak found immediately downstream of 3’ end Wider peak 1 nt downstream of 3’ end CAGE peak marks Drosha cleavage site

CAGE tags 0 position – first nucleotide downstream of 3’ end Number of tags as a function of starting position relative to pre-miRNA 3’ end CAGE peak is located one nt downstream of the 3’ end of pre-miRNAs PROVING ZEBRA FISH

Expression profile – Miru28 visualization Cell type specificity of miRNA Clustered together based on similar expression patterns Annotated Graphing program Robust mature miRNA Cell type specificity of miRNA

Validation of miRNA expression quantification -selected miRNAs that were cell type specific and broadly expressed -horizontal axis – sRNA sequencing -vertical axis – qPCR -spearman correlation average was 0.79 Expression of 12 selected broadly expressed and cell type specific miRNAs as measured by sRNA sequencing (horizontal axis) and by qPCR (vertical axis) in CD19+ B cells, dermal fibroblast, hepatocytes (each originating from three independent donors), and H9 embryonic stem cells (as a single replicate). The average Spearman correlation between the sRNA sequencing and qPCR expression quantitation across cell types was 0.79. In the qPCR measurements, Ct values were normalized to those of snoRNA SNORD48, which showed little variation between cell types (bottom panel).

Variability of miRNA expression Expression levels varied and skewed C – known and candidate miRNAs Not all miRNAs are highly expressed Average of 5 miRNAs contribute to half of the expression Most expressed at low levels Performed normalization strategies Most expressed at low levels

miRNAs in primary cell samples Depleted/elevated based on cell type Previously described in literature miRNAs depleted or elevated in cells Depleted in leukocytes and pluripotent stem cells miR-100-5p, miR-29a-3p Elevated in pluripotent stem cells, leukocytes, and hepatocytes miR-122-5p, miR-142-5p, and miR-302a-5p

Sequence conservation of promoter regions Higher conservation: Intergenic and intronic miRNAs TF genes TF = transcription factor genes – similar conservation Distance btwn TSS of pri-miRNA and mature miRNA was strongly conserved - Mouse not shown Higher than non-TF protein coding genes Average phastCons44 score miRNAs Higher conservation: Intergenic and intronic miRNAs TF genes

Distance between transcription start site and 5’ end of first pre-miRNA Genomic conservation Correlated with mouse genome (mouse not shown) Intronic and intergenic miRNAs Genomic conservation CHECK IF PRE OR PRI (MAKE SURE TO CHECK THE OTHER SIDES AND USE THEM PROPERLY!!)

Expression level – Spearman correlation Correlation higher for differentially expressed miRNAs C – correlation higher for differentially expressed miRNAs D – 11% human miRNA polycistronic (multiple) Expression at transcriptional level C – mature pri-miRNA, randomly paired pri-miRNA, and mature miRNA D – mature miRNA from same pri-miRNA and different pri-miRNA Higher correlation – differentially expressed miRNAs Correlation higher for differentially expressed miRNAs Polycistronic miRNA – 11% Transcriptional level expression

miRNA paralogs – cell type dependent expression Broad vs elevated expression Mir-128-1 Broadly expressed – primary cell samples Mir-128-2 (paralog) Elevated in brain samples

Summary of results Expression profile of miRNA, primary transcripts, and promoters Focus on primary cells miRNA promoters conserved Correlated expression profiles of mature and primary transcripts

Discussion Expanded reads from first miRNA atlas Atlas – widest range of primary cells Cell type identity sRNAs that do not meet all criteria – could belong to different RNA class Methods/tools for promoter identification – could be used for transcriptional regulation of miRNA analysis Biomarkers discovery Original atlas – 1,300 read per sRNA library, 4.4 million reads/library in Fantom5 Permissive vs robust set

References De Rie, et al. “An integrated expression atlas of miRNA and their promoter in human and mouse.” Nature Biotechnology 35, 872-878, 2017.