1. 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

2. 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

3. 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)

4. 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

5. 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

6. 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

7. 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

8. 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

9. 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

10. 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 In the qPCR measurements, Ct values were normalized to those of snoRNA SNORD48, which showed little variation between cell types (bottom panel).

11. 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

12. 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

13. 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

14. 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!!)

15. 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

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

17. 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

18. 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

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