Nucelic acids 5- Small RNAs in the regulation of biological processes Flashcards
How do we know that most of the genome is not that important
Variance in genome size suggests most of it is not that important. However, more energy is required to replicate DNA. Note the genome of the Amoeba, a single cell organism, has a genome 100 x the size of the human genome. Each amoeba contains ~ 100m of DNA. More genes does not necessarily result in a more complex organism
What is ENCODE
The Encyclopedia of DNA Elements,
a project funded by the National Human Genome Research Institute to identify all regions of transcription, transcription factor association, chromatin structure and histone modification in the human genome sequence.
This expansive resource of functional annotations is already providing new insights into the organization and regulation of our genes and genome.
What do intros code for
approximately 20% of noncoding DNA in the human genome is functional while
an additional 60% is transcribed with no known function.
Much of this functional non-coding DNA is involved in the regulation of the expression of coding genes.
Describe, simply, gene regulation
The expression of each coding gene is controlled by multiple regulatory sites located both near and distant from the gene.
Genome-wide association studies have determined that approximately 90% of single-letter differences in sequences that are associated with various diseases fall outside of protein coding regions.
Previously it was not clear how these sequence differences could influence disease however new gene regulatory sites discovered by the ENCODE project in many cases provide an explanation.
Hence, mutations in ncDNA can still result in phenotypic changes.
What is a ncRNA
Includes house keeping ncRNAs (ie ribosomal RNA, tRNA and spliceosome snRNAs)
Any RNA molecule that is not translated into protein (ie Does not include mRNA)
Also includes regulatory ncRNAs
microRNA - control the translation of perhaps most genes
siRNA / RNAi – viral defence, experimental tool
piRNA – fascinating, clearly important for germ cell production
long ncRNA:- Xist – important for X chromosome inactvation
H19 – involved in imprinting of the IGF-1 gene plus many more anti-sense RNAs with potential for imprinting and gene regulation.
What is X-Inactivation (lyonization)
The process by which one of the two copies of the X chromosome present in female cells is inactivated
The inactive X chromosome is silenced by its being packaged in such a way that it has a transcriptionally inactive structure called heterochromatin.
X-inactivation prevents female cells from having twice as many X chromosome genes as males cells, which only possess a single copy of the X chromosome
The choice of which X chromosome to inactivate is random. Once an X chromosome is inactivated it will remain inactive throughout the lifetime of the cell and it’s derivatives.
Lineage maintains inactivation of same X chromosome- hence in karyotype- one X chromosome is shorter.
How can antisense RNA be used as a tool to block RNA function
Antisense RNA binds to the RNA (mRNA)- forming a duplex, preventing the mRNA from being translated into the protein.
RNA binds more strongly to other RNA molecules than it binds to DNA.
Describe the mechanism of RNA interference
The double stranded foreign DNA is cleaved by DICER, Dicer; Rnase III-like endonuclease activity. Digests dsRNA into 21-25bp fragments of double stranded siRNAs. siRNA-guided, endonuclease activity acts to remove one of the siRNA strands, known as the “passenger strand”. Requires “Argonaute-Piwi” proteins
Multiprotein, RNA-induced silencing complexes, or “RISC” complexes formed. These recognise and cleave target mRNA molecules which have complementary sequences to the incorporated single-stranded guide siRNA
RNA interference may be the end fate of when mRNA binds to the antisense RNA
Why is RNA interference important
RNA silencing is used by plants as a defence mechanism against virus infection.
Works through the generation of small single stranded RNAs that anneal to viral RNA and cause these to degrade.
What are the features of miRNAs
Typically 18-26 nucleotides in length
Evolutionarily conserved
Ubiquitously expressed in metazoan cells
>700 miRNAs identified in humans (miRBase)
Predicted to regulate the expression of >30% of all protein-coding genes
Describe the process of miRNA transcription
Intron gene Transcribed by RNA polymerase II or III into a long primary pri-miRNA transcript with a stem-loop structure of up to several kilobases in length - pri-microRNA
Describe how pri-microRNA is processed
pry-miRNA processed by the RNase III endonuclease, Drosha, in a complex with DiGeorge syndrome critical region 8 (DGCR8). Produces 60 – 80 nucleotide precursor miRNA (pre-miRNA).
How is the pri-microRNA exported out of the nucleus
The resulting pre-miRNA is transported to the cytoplasm by exportin-5 and a Ran GTPase. Once in the cytoplasm, pre-miRNAs are processed into ~22-nucleotide duplexes by another RNAse III enzyme, Dicer, in association with TRBP. The strand corresponding to the mature miRNA is subsequently loaded onto the RISC. Mature miRNAs bind the 3’-untranslated region of target mRNAs and subsequently destabilize them- degraded by nucleases RISC, block their translation- degraded by nucleases in the cytoplasm - or both. Destruction of mRNA releases the RISC, allowing a single mi-RNA molecule to destroy lots of mRNA molecules
What features of miRNA make them useful regulators of gene expression.
A single miRNA can inhibit a whole set of different mRNAs so long as the mRNAs carry a common sequence- usually located in the 5’ or 3’ end of the untranslated regions- leading to coordinated gene regulation.
A gene that encode miRNA occupies relatively little space in the genome compared to genes that encodes a transcription regulator.
Describe how miRNAs bind to the target mRNA
miRNAs inhibit target mRNAs through base pairing with incomplete complementarity. The “Seed” region - the most important region of the miRNA for targeting. Lies between nucleotide positions 2 to 8 from the 5’ end, and is often flanked by adenosines. Mismatches, predominantly in the centre forms a bulge, often followed by a less stringent degree of complementarity in the 3’ region
