Lec 4 

• Alternative regulatory mechanisms that control gene expression
• In 1993 Scientists studying the genome of C. elegans described groundbreaking investigations relating to the non-protein coding genes of DNA
• They found that these sequences were “transcribed but not translated” 
• They showed that these primary transcripts were processed into small pieces of RNA, 18 to 30 nucleotides in length.
• They showed that these “microRNAs were able to exert control over mRNA expression levels
• Later these were shown to be relevant to all eukaryotes
• Transcription/ translation- functional
• mRNA
• hnRNA
• tRNA
• rRNA
• Transcription/ translation – most regulatory (bar snRNA and snorna, others are forms of RNAi (RNA interference)
• Small RNA’s
• snRNA
• miRNA
• siRNA
• snoRNA

Intron splicing process – using snRNA

• In higher eukaryotes, nearly all genes contain intron sequences that must be spliced out of pre-mRNA to form mature mRNA species.
• Pre – mRNA splice site consensus sequences located at the extreme ends of introns help direct splicing reactions
• The GU di- nucleotide at the 5’ splice site of the intron and the AG di- nucleotide at the 3’ splice site are highly conserved

SnRNA’s reminder

• Small nuclear RNA’s
• Involved in splicing of introns
• Only rarely do introns self- splice
• Part of ‘spliceosome complex’
• The spliceosome (mass similar to ribosome) is responsible for recognising and cutting the introns
• Made up of 5 small nuclear ribonuclear subunits + > 100 proteins
• snRNP’s are small ribonucleoprotein particles each containing snRNA’s that are U-rich (hence named U1, U2 to U6 etc) that act within the Spliceosome complex.

How do SnRNA’s function?

• They are assembled from the 5 splicing snRNP’s (snurps) and their pre- mRNA
• Interactions between the U1 snRNA at the 5’ splice site and the U2 snRNA at the branch point sequence (called branch point A) are crucial in selecting where splicing occurs

Cytoplasmic mechanisms of Post- transcriptional control

• Until recently, regulation of gene expression was traditionally believed to occur predominantly through transcriptional control
• However, there are mechanisms that operate in the cell regulating mRNA through other means
• mRNAs can be highly stable and can exist in high concentrations within the cytoplasm (many prokaryotic genes)
• mRNA’s can also be fleeting in their existence (more the case for eukaryotes) where bursts of protein expression are required
• Translational silencing is an important control point

Transient Duration of RNA

• In transient cases, mRNA existence is predominantly controlled by degradative mechanisms
• The primary determinants of stability are the cap and the tail
• Either one or both of these must be removed for mRNA degradation to begin or the mRNA must be cleaved internally
• there are 3 possible pathways of degradation

3 pathways of mRNA degradation

1. Deadenylation - dependent mRNA decay

• MOST mRNAs undergo decay by this pathway.
• Here, the polyA tail is gradually shortened by deadenylase activity.
• mRNAs are normally degraded by the CCR4 (exonuclease) dependant mechanism.
• Some mRNA’s are targeted for more rapid degradation via the additional use of special factors.
• The degradation machinery of the cytoplasm is attracted to the deadenylated mRNA and rapidly degrades the mRNA in either the 5’ to 3’ direction or in the 3’ to 5’ direction by one of two mechanisms
• 5′→3′ decay via the Lsm1–7 complex which associates with the 3′ end of the mRNA transcript and induces decapping by the DCP1–DCP2 complex leaving the mRNA susceptible to decay by the 5′→3′ exoribonuclease XRN1
• 3′→5′decay, by the exosome complex (a multi-protein intracellular complex capable of degrading various types of RNA). This is followed by decapping by the scavenger-decapping enzyme DcpS

2. Deadenylation – independent mRNA decay

• Although most transcripts undergo deadenylation-dependent decay, there are several exceptions. Certain mRNAs seem to bypass the standard pathways ….
• In Saccharomyces cerevisiae, deadenylation-independent pathways require recruitment of the decapping machinery as the tail is blocked to deadenylase activity
• Here, Rps28B interacts with an enhancer of decapping-3 (Edc3) to engage the decapping enzyme (DCP1/2).
• Following decapping, the mRNA is degraded by XRN1.

3. Endonuclease mediated mRNA decay

• Endonuclease-mediated mRNA decay initiates with internal cleavage of the mRNA, which generates two fragments each with one unprotected end (note these are not Ribozymes).
• The fragments are degraded by XRN1 and the exosome.
• This is perhaps the most efficient means of destroying mRNAs and several cellular endonucleases that target mRNA have been found (even nucleolar rRNA endonucleases) though many are undiscovered.

Mi RNA – micro RNA

• Ss RNA fragments approximately 21 – 25 nucleotides long
• Function as post- transcriptional regulators and thought to target 50 – 60% of mammalian genes
• Most are cellular (some are circulating) and functional in RNA silencing through base- pairing with complementary sequences in the mRNA molecule
• Base pairing in plants is near- perfect.
• Base pairing in animals only requires 6- 8 nucleotides that act as a ‘seed’
• Through base- pairing, the mRNA is then either:
• Cut
• Destabilised by shortening of the polhyA tail (deadenylation)
• Translation from the mRNA is repressed

Synthesis of miRNA in animals

• Genes for miRNA are transcribed to a primary miRNA (pri- miRNA)
• The pri- miRNA is processed  within the nucleus to a precursor miRNA (pri- miRNA) by Drosha, a class 2 RNase III enzyme to become a pre- miRNA (hairpin structure)
• Next, the transport of pre- miRNA to the cytoplasm is mediated by exportin- 5 (EXP-5)
• In the cytoplasm, they (70bp) are further processed to become mature miRNAs (21- 25bp) by Dicer an RNase III type protein that cleaves the miRNA & removes the Hairpin
• Finally the miRNA is loaded onto the Argonaute (AGO2) protein which removes a single strand to produce the effector RNA-induced silencing complex (RISC) (or miRISC once the miRNA is bound)

Synthesis of miRNA in plants

• The 2 step processing of pri- miRNA into mature miRNA occurs entirely in the nucleus and is carried out by a single RNase III enzyme
• DCL1 (Dicer- like 1)
• The mature miRNA are then bound by argonaute subfamily proteins

Action of miRNA

What decides whether they will remove the mRNA or regulate translation?

• Gene silencing may occur either via mRNA degradation or preventing mRNA from being translated. The degree of miRNA – mRNA complementarity is a major determinant of the regulatory mechanism process
• Complete complementarity: Ago2 can cleave a specific mRNA and lead to direct mRNA degradation mechanisms via deadenylation, decapping, and exonuclolytic digestion
• Incomplete complementarity: if miRNA bind to the 3’ UTR regions (3’ untranslated region) of complementary mRNAs via imperfect base- pairing they repress translation, often of more than one mRNA! The exact mechanism  for the repression of target mRNA translation by miRISC is still unknown (initiation or elongation possibly)

miRNA relevance to life

• miRNAs have key roles in the regulation of distinct processes in mammals and are therefore therapeutic targets.
• They are conserved in the roundworm Caenorhabditis elegans (a notable model organism) and some are known to have a role in development, since loss-of function mutations are larvae-lethal.
• Some members are involved in adipocyte differentiation and even fat metabolism
• They are also involved in late stage limb development through regulation of Hox genes…(see other lecture)
• Harmful genes could be suppressed through the introduction of synthetic miRNAs into diseased tissues in an attempt to restore normal cellular functions that have been affected by the mis- regulation of one or more miRNA
• In contrast, some researchers have utilized miRNA inhibitors (steric blockers) in an effort to increase the endogenous levels of therapeutic proteins.

siRNA (small interfering RNAs)

• siRNA is a short single stranded ds oligomer of 21 – 22 bps
• siRNA also downregulate gene – expression guided by sequence complementarity and can be used therapeutically to block the synthesis of disease – causing proteins
• the big difference between them lies in function. Whereas miRNAs act as regulators of endogenous genes, siRNA acts as defenders of genome integrity in response to foreign or invasive nucleic acids such as viruses (innate immunity) and also plays a role in chromatin structure
• siRISC can direct heterochromatin formation by associating with nascent transcripts and RNA polymerases which can lead to the association or activation of a DNA methyltransferase (DMT) that methylates the DNA, leading to heterochromatin formation

SiRNA synthesis

• process is similar to that of miRNA
• many RNA structures can be processed by dicer to produce siRNAs, even viral DNA
• siRNA is bound by Ago and then binds RISC to form a complex
• the siRNA guide strand directs RISC to perfectly complementary RNA targets, which are then degraded

miRNA and siRNA Comparison

• Differences
• siRNA externally sources (viral) miRNA internally sourced (cellular)
• siRNA has very high to 100% complementarity in animals, miRNA only requires a ‘seed’ of 6-8 nucleotides. Therefore, gene targeting of siRNA is specific (one gene) whereas miRNA is broad (many)
• siRNA more likely to bring about cleavage than miRNA in animal cells
• similarities
• use the same processing machinery within the cell
• act through the same mechanisms to bring about mRNA degradation

• in general, the major difference between them is that siRNA is specific to a particular target (gene sequence) whereas miRNA is not and a single miRNA can potentially regulate the expression of many different genes
• however, recent papers seem to imply that there are miRNAs and siRNAs with intermediate states and we should be focusing on the RNAi mechanism (interference RNA)

Why use them for therapeutics

• highly potent
• 2 MOAs
• Introduction of a systemic miRNA or siRNA into target cells to illicit RNA interference
• Synthetic ssRNAs targeting miRNA by acting as miRNA antagonists (anti – sense therapy)
• Synthetic miRNA being used to replace faulty miRNA in a replacement- type approach

Gene- slicing efficiency

• Increasing the length of the double stranded RNAs make them more potent (u to 100x). In this form they are acted on by the dicer and are referred to as ‘dicer ready’ or ‘dicer substrate’ and are loaded onto RISC more efficiently
• Efficiency varies depending on the region of the mRNA to which they are targeted
• Targeting the guide strand is key
• Reducing siRNA concentration to the minimum found to work to prevent off – target cutting
• Some siRNA have miRNA like effects therefore miRNA seed sequences must be avoided
• Using multiple siRNAs against same mRNA ensures increased efficiency

Piwi RNA’s

• One of the most abundant small RNAs in the mammalian cells
• Larger than miRNAs (21 – 35 nucleotides long)
• Not as conserved as miRNAs and much more complex
• Form piRNA protein complexes (dicer independent)
• Function in gene silencing of transposable elements (jumping genes)
• Organisms evolved mechanisms to regulate the mobilization of transposable elements and to maintain genome integrity since transposon movement can induce mutations 
• one mechanism is by packing transposon DNA into heterochromatin and methylating the histones. Histone methylation recruits proteins that induce transcriptional silencing piRNA is involved in regulating chromatin architecture via methylation processes
• another is through the piRNA: piwiprotein complex that silence transposable elements by forming RISC complexes and cleaving transposon and retrotransposon RNA
• piwi RNA are especially important in testes where they prevent transpositions in the germ- cells that can be passed onto the next generation

Bacterial small RNA (sRNAs)

• Amount to hundreds not thousands
• Anti- sense sRNAs: the most prevalent role for antisense sRNAs in bacteria has been the repression of genes that encode potentially toxic proteins
• Base pairing sRNAs with limited complementarity: bacterial version of miRNAs and siRNAs
• They modulate mRNA stability and translation
• However, are generated as unprocessed single entities (100 bases long approximately)
• Requires at least a seed region of 6- 8 contiguous base pairs and regulatory outcomes many
• Some sRNAs are bifunctional in that they can also code for proteins or function as ribozymes

snoRNAs – small nucleolar RNA

• RNAs that are only found in the nucleolus as the name suggests 60 – 300 bases long
• Guide site – specific post transcriptional modification of pre – rRNA, pre- tRNA and pre- snRNAs.  Found from Archea all the way up to mammals
• The post translational modifications of rRNA must be important for protein transcription  because the modified bases all occur at highly conserved core regions
• Two main classes of snoRNAs have been defined
• Box C/D snoRNAs
• Box H/ ACA snoRNAs
• Differ in sequence, structural elements, binding partners and nature of modification catalysed
• Note: a number of orphan snoRNAs have also been found with no apparent function

Function of snoRNAs

• The positions of 2’0 ribose methylation and pseudo uridine formation are determined by about 150 of these nucleolus- restricted RNA species
• Box C/D snoRNAs – associated with methylation (methyl transferase)
• Box H/ ACA snoRNAs – associated with pseudo uridylation
• The post- translational modification mechanisms require transient hybridization
• Different snoRNAs direct these modifications at specific sites of the pre- RNA
• In order to act the snoRNAs must bind snoRNPs
• Other functions of snoRNAs
• Note other modifications can occur to pre- rRNA not involving snoRNAs
• Some snoRNAs have been shown to have a function in alternative slicing by generating shorter RNAs
• A number of small RNAs with evolutionary conserved position and size are derived from snoRNAs and some of these are capable of binding AG02 implying they have a function in gene regulation

Other RNAs

• scaRNAs

small cajal body – specific RNAs. These are a subtype of snoRNAs found in the cajal body (an organelle found in the nucelus of clls that acts as a concentrating body for RNA processing). scaRNA functions in the synthesis of snurps, guiding modifications (methylation and pseudouridylation) of the snaRNAs U1, U2, U4, U5, and U12

Roboswitches

• bacterial alternative to transcriptional regulation such as occurs in operons and as a mechanism is widespread in bacteria
• they are RNA elements encoded within a mRNA that directly affect the expression of genes encoded in the full transcript (in other words they are cis- acting elements but in an RNA context)
• these regulatory elements (known as riboswitches) are specifically defined as:
• mRNA elements that bind metabolites (or metal ions) as ligands  and regular mRNA expressions by forming alternative structures in response to this

Location and Example

• most riboswitches are present in the 5’ UTR of the mRNA preceding the start codon in bacterial mRNA. Riboswitches regulate the transcription or translation of the mRNAs of which they are a part by being sensitive to metabolite concentration
• The classic example is Bacillis subtillis xpt pbuX ‘operon’ which encodes enzymes involved in purine synthesis
• In Bacillus subtillis, this mRNA motif is located on at least five separate transcriptional units that together encode 17 genes involved in purine transport and biosynthesis
• If the metabolites are low (-M), the aptamer will allow transcription read- through. High concentrations of the metabolite (M+) induce transcription termination
• Riboswitches are composed of 2 domains
• The aptamer domain
• (ligand- binding domain) which is a complex tertiary structure brought about by the folding of the RNA. The aptamer domain acts as a specific receptor that binds a specific ligand/ metabolite when it reaches a high enough concentration
• The expression platform
• Regulates gene expression through its ability to toggle between 2 different secondary structures in response to ligand binding
• common to both domains is something called the switch sequence (that can pair with both the aptamer domain or the expression platform)
• its placement (pairing) in the aptamer domain or the expression platform ultimately dictates the expression outcome of the mRNA

Aptamers

• many studies have now confirmed that the complex 3 dimensional shapes that some RNA molecules adopt can mimic proteins receptors and anti – bodies in their ability to selectively bind proteins or small molecules
• ligand – binding induces conformational change and modulates gene expression
• Translational riboswitches control gene expression by regulating the accessibility of the ribosome binding site or start codon
• Transcription – regulating riboswitches lead to the formation of a ρ- independent terminator, which inhibits elongation by destabilizing the RNA: RNA polymerase complex
• Other aptamers act through inducing mRNA degradation, alternative 3’ end processing of mRNA or through an intrinsic ribozyme activity in response to ligand binding