miRNA / RBPs / Transcriptome

			Contain AU-rich elements (AREs). Proteins bind AREs to affect the stability or decay rate of transcripts in a localized manner or affect translation initiation.
			The 3'-UTR also has silencer regions which bind to repressor proteins and will inhibit the expression of the mRNA.

• miRNA
  • definition
    • is a small non-coding RNA molecule (containing about 22 nucleotides)
  • How many mi RNA
    • The human genome may encode over 1900 miRNAs,[6] although more recent analysis indicates that the number is closer to 600.[7]
  • Structure
  • miRNA gene
    • location
      • As many as 40% of miRNA genes may lie in the introns or even exons of other genes.[42]
  • Target
    • target about 60% of the genes of humans
    • Targeting
      • A given miRNA may have hundreds of different mRNA targets, (on average 400)
      • a given target might be regulated by multiple miRNAs.[
  • Expression
    • miRNAs are abundant in many mammalian cell types[
  • Nomenclature
    • “miR-” refers to the mature form of the miRNA,
    • “mir-” refers to the pre-miRNA and the pri-miRNA
    • The miRNAs encoding genes are also named using the same three letter prefix
  • Biogenesis of mi RNA

Transcription
RNA polymerase II (Pol II).[47][48] binds to a promoter found near the DNA sequence→ encodes the hairpin loop of the pre-miRNA	initially transcribed as part of one arm of an ~80 nucleotide RNA stem-loop→ that in turn forms part of a several hundred nucleotide-long miRNA precursor termed a primary miRNA (pri-miRNA).[	→ capped with a specially modified nucleotide at the 5' end, polyadenylated with multiple adenosines (a poly(A) tail),[47][43] and spliced. → pre-miRNA로 됨	pre-miRNA exits the nucleus	In the cytoplasm, the pre-miRNA hairpin is cleaved by the RNase III enzyme Dicer., yielding an imperfect miRNA:miRNA* duplex about 22 nucleotides in length→ duplex중 한 strand가 incorporated into the RNAinduced silencing complex (RISC) where the miRNA and its mRNA target interact.

• function

recognize their target mRNAs by using as few as 6-8 nucleotides (the seed region) at the 5' end of the miRNA,[

base-pairing with complementary sequences within mRNA molecules (주로 3'UTR region 에 결합)

Target mRNA silincing (1) Cleavage of the mRNA strand into two pieces (2) Destabilization of the mRNA through shortening of its poly(A) tail ('m RNA degradation) (3) Less efficient translation of the mRNA into proteins by ribosomes.[eg. miRNAs occasionally also cause histone modification and DNA methylation of promoter sites,)

End result ↓ translation of protein

• MicroRNAs in Biological Fluids
  • Definition
    • MicroRNAs are non-coding genomic regions, around 20 nucleotides long, that control gene expression through transcription silencing
  • MicroRNAs in Biological Fluids
    • extracellular miRNAs are highly stable
    • Two populations of extracellular miRNAs exist in biological fluids.
• One can be found in vesicles such as exosomes, microvesicles, and apoptotic bodies (116, 120)
  • the other is associated with proteins, especially AGO2, HDL) (126, 127) and nucleophosmin
• Database of miRNA
  • http://mirbase.org

RNA binding proteins (RBPs)

• definition
  • )
• How many mi RNA

  • 800 RBPs]

• Structure
• Function

		Results (다음 셋 변화)
		splicing,
		abundance
		location within the cell.

Transcriptome

• Definition
  • a collection of all the gene readouts present in a cell.
  • the entire collection of RNA sequences in a cell
• What can a transcriptome tell us?
  • when and where each gene is turned on or off in the cells and tissues of an organism (as different cells show different patterns of gene expression)
  • the number of transcripts 알아서, → to determine the amount of gene activity - also called gene expression - in a certain cell or tissue type.
  • Xx cell에서는/xx병에서는 xx gene 이 많이 발현하고/active하고/역할하고 있구나.
  • 한 마디로 ‘Gene Expression Profile’
• Database of transcriptome
  • the National Human Genome Research Institute (NHGRI),
    • which is part of the National Institutes of Health (NIH)
    • Projects
      • The Mammalian Gene Collection initiative
        • a free, public library of human, mouse, and rat mRNA sequences.
      • The Mouse Transcriptome Project
        • a free, public database of gene transcripts for many mouse tissues.
        • Link: Mouse Reference Transcriptome Database
  • the Genotype-Tissue Expression Project (GTEx) : Arthur
    • https://gtexportal.org/home/
    • a catalog of human gene expression in a variety of different tissues,
    • gene expression, exon expression, eQTL
  • the Encyclopedia of DNA Elements (ENCODE).
    • aim to characterize and understand the working parts of the genome,
  • Novartis
  • Genevisible: https://genevisible.com/tissues/HS/Gene%20Symbol/S100A10
  • the European Molecular Biology Laboratory

Gene Expression Analysis (Transcriptome)

Methods comparison

sample preparation

Frozen	Formalin-fixed paraffin embedded (FFPE)	Formalin
suitable	Perfectly adequate for genomic analysis and comparable to frozen tissues	Not good, because it causes cross linking of proteins and nucleic acids to each other: most nucleic acide are degraded (to lengthes of 200-300 base pair)

The average mRNA concentration is 10⁻⁴–10² copies/cell

Interpretation

		principle	Protocol		Readout
PCR		amplification of DNA by polymerase chain reaction (PCR) is monitored in real time.		amplified DNA 를 봄, TaqMan (hydrolysis이용) 이 q PCR에 최적화된 assay 방법임.	the lowest quantification limits and the least biased results, in comparison to microarrays or RNA-seq., low throughput, not amenable to perform global expression analyses
microarray= DNA chip, biochip, gene-array				Spot 준비: membrane). The spots can be DNA, cDNA , or oligonucleotides. ☐ The hybridization of an mRNA molecule to the DNA template from which it is originated. -- ☐ Spots with more intensity are obtained for diseased tissue gene if the gene is over expressed in the diseased condition	rapidly being replaced by sequencing technologies
RNAseq		poly(A) capture,: a widely used protocol: (in both RNA-seq and microarray analyses) to restrict the analysis to mature mRNA [20, 30, 46].		Caveat: However, this library preparation method only picks up RNA fragments with a poly-A tail, introducing substantial bias in low quality RNA samples
	Whole-transcriptome sequencing (RNA-seq) technology has emerged as a revolutionary platform for genome-wide quantification of mRNA transcripts. This technique enables the	NGS, broader dynamic range, to examine DNA variations (SNPs, insertions, deletions) and even discover new genes or alternative splice variations using just one dataset Hirozane san 은 이것이 quantitative방법은 아니라고 하네.	active ribosomal RNA (rRNA) depletion and capture by random primers, such as the	RNA-seq requires a significant amount of starting material, it will not be applicable when only small amount of RNA is available	Example: WORLDSymposium 2021-Perez (GBA-PD mice model)
					TPM: Transcripts Per Million ,unit of transcript expression TPM) is a normalization method for RNA-seq, should be read as "for every 1,000,000 RNA molecules in the

Uncertain Spans

location	text/status	reason
Top continuation table	row count	Only the bottom edge of the prior photo’s mRNA/UTR table is visible; the upper rows are not in this crop.
function table	Target mRNA silincing	Spelled with an i instead of e in the source heading; preserved as written.
Interpretation table / RNAseq Readout last row	last sentence cut off	The bottom RNA-seq Readout cell (“TPM … for every 1,000,000 RNA molecules in the …”) is cut off at the bottom edge.