Small RNAs in the regulation of biological processes Flashcards
What is the complexity of an organism related to?
- not necessarily related to genome size (C value) b/c constant amount of genetic material from same species
What was found through ENCODE?
- 20% of non coding DNA functionally active - involved in gene regulation (involved in regulation of gene expression)
- 60% transcribed with no known function
- 5% of genome conserved, nearly all protein coding genes and substantial number of genes for noncoding RNAs
- 95% is junk DNA, but still transcriptionally active
Define non coding RNAs:
- any RNA molecule not translated into protein (excluding mRNA)
What are the types of non coding RNAs?
- miRNA: control translation of most genes
- housekeeping ncRNA (rRNA, tRNA, splicesosome snRNA) - splicing and other RNA processing reactions
- siRNA (silencing) - inhibit expression / RNAi (interference) viral defense and experimental tool
- piRNA (piwi interacting) - germ cell productions, form RNA protein complex via interactions with piwi proteins (regulatory proteins repsonsible for stem and germ cell differentiation), epigenetic and post-transcriptional gene silencing
- long ncRNA - important for X chromosome inactivation (H19) , imprinting IGF-1 and many more antisense RNA with potential for imprinting and gene regulation
What occurs during X chromosome inactivation?
- Xist (long ncRNA) controls it
- Packaged in such way that has transcriptionally inactive structure (heterochromatin)
- Prevents females having twice as many X chromosomes genes as males
- Randomly selected, but will remain inactive for life
How does one block a gene?
- transcribe reverse of genes to make antisense RNA strand (form h-bonds so double stranded so no transcription) to produce dsRNA
- dsRNA broken down into fragments (siRNA) and then used to target inhibition of gene expression i.e dsRNA derived from virus and used to produce siRNA
How does RNAi work?
- DICER breaks up dsRNA into 21-25bp fragments via RNAase III like endonuclease activity
- Passenger strand (1 of siRNA strands) removed by siRNA guided endonuclease activity
- Requires “Argonaute piwi proteins” (AGO)
- RISC (multiprotein RNA silencing complex) created - recognizes + cleaves target mRNA molecules with complementary sequences to incorporated single strand guide siRNA
What occurs if DICER is not present?
- Without DICER lethality in embryonic stage because stem cells unable to differentiate so depletion of multipotent stem cells causing
- Incomplete embryonic myogenesis
- Limb morphogenesis defect
- Lung development defect
- Epidermal hyperproliferation
What are microRNAs?
- genomically encoded snRNA in gene regulation - occurs naturally in genes
- mRNA present in genome involved in regulating other genes so more organization levels
- controls translation of most genes
How is miRNA synthesized?
- made as long primary pri-miRNA transcript (can be several thousand bases long) - folds back on itself to form a stem-loop structure (extended region of dsRNA) due to sequence characteristics
- Processing in the nucleus gives shorter pre-miRNA 70 nucleotides long
- Exported from the nucleus (actual final product made in cytoplasm)
- Offered to DICER RNAase with same processing pathways to create miRNA which can act as RNAi with RISC complex on this
- Modulation of gene expression and cause gene knockdown (dec. In protein amount from gene)
What is the structure of miRNA?
- 2 matching areas separated by bulge: match-bulge-match
○ bulge has less stringent degree of complementary in 3’ region
○ can fold over to produce snRNA
What is the seed region?
- between nucleotides 2-8 from 5’ end, flanked by adenosine - most important in targeting bioinformatics in gene location, to see if have heridatory disease
- Each miRNA can target several different mRNAs leading to coordinated gene regulation
What are most miRNA target site?
- within 3’UTR - position and number of binding sites influence repression efficiency
UTR: untranslated region
What human diseases have faulty miRNA?
- Expressed in leukemia, carcinoma, heart disease
Chronic Lymphoid Leukemia: deletion of part of gene on Chr14 causing miRNA loss and promoting CLL
Drosha:
RNAse initiating the processing of microRNA (miRNA), or short RNA molecules from their Pri-miRNA precursor transcripts.
in the cell nucleus.
How does RISC work?
incorporates one strand of an siRNA or miRNA and uses this as a template for recognising a complementary mRNA target.
When the complementary target strand is bound, the RISC RNase is activated, leading to cleavage of the target RNA.
