Ch.11: Non-Coding RNAs

2 benefits of RNA interference

-This mechanism represents an important form of regulation. When genes encoding pri-miRNAs are turned on, the production of miRNAs silences the expression of specific mRNAs. - RNA interference provides a defense against viruses. This mechanism is widely used in plants to prevent viral infections

Key Roles of SRP RNA

1. SRP RNA provides a scaffold for the binding of SRP proteins 2. After SRP binds to the SRP receptor in the ER membrane, the SRP RNA stimulates proteins within both SRP & the SRP receptor to hydrolyze GTP. SRP RNA alters the structures of these proteins to enhance their GTPase activities. This stimulation is essential for the release of SRP.

After binding to an mRNA

2 things may happen: - RISC may inhibit translation w/o degrading the mRNA. This is more common for miRNAs, which often are only partially complementary to their target mRNAs - RISC may directs the degredation of the mRNA. 1 of the proteins in RISC can cleave the mRNA. This usually occurs for siRNAs that typically are a perfect match (or highly complementary) to their target mRNA. *These 2 effects are termed RNA interference bc the miRNA or siRNA interferes w/ the proper expression of mRNA.

IPSI

Affect the ability of plants to cope w/ phosphate starvation

miR402

Affects the rate of seed germination & seedling growth under stress conditions.

Crispr Interference

After the tracrRNA-crRNA-Cas9 complex has formed, the bacterial cell is ready to destroy the bacteriophage DNA. This phase is called interference bc it resembles the process of RNA interference. Each spacer within a crRNA is complementary to one of the strands of a bacteriophage DNA. Therefore, the crRNA acts as a guide that causes the tracrRNA-crRNA-Cas9 complex to bind to that bacteriophage DNA. After binding, the Cas 9 protein functions as an endonuclease that makes double-strand breaks in the bacteriophage DNA. This cleavage inactivates the phage & thereby prevents phage proliferation.

HOTAIR Diseases

Certain types of cancer, such as breast cancer, may occur when HOTAIR isn't functioning properly.

Telomere shortening

DNA polymerase synthesizes DNA in a 5' to 3' direction & requires a primer. For these reasons, DNA polymerase cannot copy the 3' ends of double-stranded DNA. Therefore, if this replication problem were not overcome, a linear chromosome would become shorter w/ each round of DNA replication.

ncRNAs & Plant health

Discoveries on ncRNAs effect on plant health will have a great impact on agriculture

ncRNA guide

Due to multiple binding sites, ncRNAs may guide 1 molecule to a specific location in a cell. An ncRNA may bind to a protein & guide it to a target site in the DNA that is part of a particular gene. This occurs bc the ncRNA has a binding site for the protein & another for DNA.

snoRNA Ribonucleoprotein (snoRNP)

First, a snoRNA acts as a scaffold for the binding of several proteins to form a snoRNP. The structure of a snoRNP can methylate ribose within rRNAs. The snoRNP contains several proteins, including 2 proteins that catalyze the methylation reaction. After snoRNP has formed, the 2nd role of snoRNA is to act as a guide. A snoRNA has antisense sequences that are complementary to sites in rRNAs. This enables a snoRNA to recognize & bind to rRNAs. The snoRNA guides the snoRNP to rRNA. Once an rRNA binds to the antisense sequence in the snoRNA, the proteins within snoRNP catalyze the modification of the rRNA.

2. Polymerization

Following biding, TERC has a 2nd function. This ncRNA has a sequence that functions as a template for the synthesis of a 6-nucleotide sequence at the end of the DNA strand. This synthesis is called polymerization, bc its analogous to the function of DNA polymerase. Telomere lengthening is catalyzed by a protein within telomerase called telomerase reverse transcriptase (TERT). TERT's name indicates that it catalyzes the reverse of transcription; it uses an RNA template to synthesize DNA.

3. Translocation

Following polymerization, telomerase then moves a process called translocation to the new end of the DNA strand Y attaches another 6-nucleotides to the end. This binding-polymerization-translocation cycle occurs many times in a row, thereby greatly lengthening the 3' end of the DNA strand in the telomere. This lengthening provides an upstream site for an RNA primer to be made. DNA polymerase then synthesizes the complementary DNA strand. In this way, the progressive shortening of eukaryotic chromosomes is prevented.

Signal Recognition Particle (SRP)

For proteins to be secreted from the cell, they are first targeted to the plasma membrane in bacteria & archea, or to the ER membrane in eukaryotes. This targeting process is facilitated by SRP an RNA-protein complex. SRP is composed of 1 ncRNA & 1 protein in bacteria. In eukaryotes, SRP is composed of 1 ncRNA & 6 different proteins.

HOTAIR scaffolding

HOTAIR acts as a scaffold for the binding of 2 protein complexes that covalently modify histone proteins. One of them binds to the 5' end of HOTAIR, & the other binds to the 3' end. HOTAIR then guides these complexes to a target gene by binding to a region near the gene that contains many purines, called a GA-rich region. HOTAIR binds to a GA-region next to a HoxD gene. A portion of HOTAIR is complementary to this GA-rich region.

CRISPR Expression

If a bacterial cell has already been adapted to a bacteriophage, a subsequent bacteriophage infection will result in the expression phase in which the system gets ready for action by expressing the Crispr, tracr, & Cas 9 genes. The Crispr gene is transcribed from a single promoter & produces a long ncRNA called pre-crRNA, which contains several repeat sequences w/ spacers in between. The gene encoding the tracr RNA is also transcribed, which produces many molecules of tracrRNA. A region of tracrRNA is complementary to the repeat sequences of the pre-crRNA. The pre-crRNA is then cleaved into many small molecules, now called crRNA. Each crRNA is attached to a tracrRNA. A region of the tracrRNA is recognized by the Cas 9 protein. The tracr RNA acts as a guide that causes the tracRNA-crRNA complex to bind to a Cas 9 protein.

RNA-induced silencing complex (RISC)

In the cytosol, both pre-miRNAs & pre-siRNAs are cut by an endonuclease called dicer. This releases a double-stranded RNA molecule typically 20-25 bp long . This double-stranded RNA associates w/ proteins to form a complex RISC. One of the RNA strands are degraded. The remaining single-stranded miRNA or siRNA is complementary to specific mRNAs that will be silenced. The miRNAs or siRNAs act as a guide that causes RISC to recognize & bind to such mRNA molecules.

ncRNA & neurological disorders

Many miRNAs are essential for the proper development & functioning of the nervous system. Appx. 70% of all miRNAs are expressed in the brain, & many of them are specific to neurons. miRNAs are involved in neuron growth, & development of the nervous system. Abnormal levels of miRNA expression are associated w/ all neurological disorders. In lzhimers, abnormal expression is though to be involved down-regulating the expression of the enzyme β-secretase, which leads to the overproduction of certain β-amyloid peptides- a key feature of the disease. miRNAs also control the inflammation that leads to sclerosis.

Telomerase RNA component (TERC)

Plays a role in DNA replication. TERC facilitates the binding of telomerase to the telomere & acts as a template for DNA replication.

small nucleolar RNA (snoRNA)

Plays a role in RNA modification, protein targeting, & secretion. A snoRNA facilitates covalent modifications to rRNAs.

Hox transcript antisense intergenic RNA (HOTAIR)

Plays a role in chromatin structure & transcription. HOTAIR alters chromatin structure & thereby represses transcription by guiding histone-modifying complexes to target genes. The gene that encodes HOTAIR is located within a cluster of genes called the HoxC genes. It is transcribed from the opposite (antisense) strand w/ respect to the HoxC genes.

X inactive specific transcript (Xist RNA)

Plays a role in chromatin structure & transcription. Xist RNA coats one of the X chromosomes in female mammals & plays a role in its compaction & inactivation.

CRISPR RNA (crRNA)

Plays a role in genome defense. crRNA, found in bacteria & archea , guides an endonuclease to foreign DNA, such as the DNA of a bacteriophage.

RNA component of signal recognition particle (SRP-RNA)

Plays a role in protein targeting & secretion. In bacteria, SRP directs the synthesis of some polypeptides to the plasma membrane. In eukaryotes, it directs polypeptide synthesis to the endoplasmic reticulum.

miR167 & miR397

Plays a role in seed development.

miR824

Plays a role in the development of stomata

microRNA (miRNA) / small interfering RNA (siRNA)

Plays a role in translation & RNA degeneration. miRNAs & siRNAs regulate the expression & degradation of mRNAs.

Ribosomal RNA (rRNA)

Plays a role in translation. rRNAs are components of ribosomes, which are the site of polypeptide synthesis.

Transfer RNA (tRNA)

Plays a role in translation. tRNA molecules recognize mRNA codons during translation & carry the appropriate amino acid.

non-coding RNA's (ncRNAs)

RNA molecules that don't encode polypeptides. ncRNA's perform a spectacular array of cellular functions in bacteria, archea, protist, fungi, plants, & animals. They play an important role in DNA replication, chromatin modification, transcription, translation, & genome defense. Only 20% of transcription involves the production of mRNAs, 80% is associated w/ making ncRNAs. Abnormalities in ncRNAs are associated w/ cancer, neurological disorders, cardiovascular diseases, etc. They are also critical in the growth of plants.

Ribozyme

RNA molecules w/ catalytic function. Ex. rRNA within peptidyltransferase catalyzes peptide bond formation during translation.

1. Binding of Telomerase

TERC has a sequence that is complementary to the DNA repeat sequence. In this way, TERC acts as a guide that allows telomerase to bind to the 3' overhang region of the telomere.

Silencing of mRNAs

The miRNA is first synthesized as a pri-miRNA (primary-miRNA) in the nucleus. Due to complementary base pairing, the pri-miRNA folds into a stem-loop w/ long, single stranded 5' & 3' ends. The pri-miRNA is cleaved at both ends to form a pre-miRNA (precursor-miRNA). The pre-miRNA is then exported from the nucleus. siRNAs don't go through the processing events that occur in the nucleus. Instead, pre-siRNAs are derived from viral RNAs, or made by researchers. The pre-siRNA is formed from 2 complementary RNA molecules that base-pair w/ each other.

CRISPR-Cas system function

The repeats within the Crispr gene are interspaced by short, unique sequences, called spacers. The CRISPR-Cas type II system also employs a gene that encodes an ncRNA called tracrRNA & a few protein-encoding CRISPR-associated genes (Cas genes), which are usually adjacent to the Crispr gene. These genes are needed to mediate the defense against bacteriophages. The CRISPR-Cas systems is considered an adaptive defense system bc a bacterial cell must first be exposed to an agent, such as a bacteriophage, to elicit a response. The defense mechanism occurs in 3 phases; Adaptation, Expression, Interference.

ncRNAs & Cancer

The roles of ncRNAs in cancer have been most thoroughly studied w/ respect to miRNAs. In some cases, the genes behave as tumor-supressor genes, bc a lower level of expression of particular miRNAs allows tumor growth. In other cases, the genes that encode certain miRNAs act as oncogenes; their over expression promotes cancer. The miRNA-200 family plays an essential role in tumor suppression by inhibiting metastasis- the process by which cancer cells can spread through the blood stream to other parts of the body. Low levels of expression of miR-200 members have been associated w/ many types of cancer; bladder, melanoma, stomach, & colorectal. HOTAIR is also highly expressed in a variety of cancers; breast, lung, & colorectal. When over-expressed, HOTAIR behaves as an oncogene. High levels of HOTAIR expression in primary breast tumors are a predictor of metastasis & death. HOTAIR is known to interact w/ a variety of cellular components, but the mechanism by which it promotes cancer isn't well understood.

# of protein encoding genes and ncRNAs genes

There are 22,000 Protein-Encoding Genes, the # of genes that specify ncRNAs is difficult to measure. Estimates range from several thousand to tens of thousands.

ncRNA Mutations

When certain ncRNAs are expressed abnormally, that is, at too high or too low of a level, disease conditions are known to occur. Abnormal expression levels can be caused by mutations in specific genes or by epigenetic changes, that alter the expression of genes that encode ncRNAs.

Alteration of protein function or stability

When it binds to a protein, an ncRNA can alter that proteins structure, which in turn can have a variety of effects: -ability of the protein to act as a catalyst -ability of the protein to bind to other molecules (Proteins, DNA, or RNA) -Stability of the protein

Blocker

an ncRNA may physically prevent or block a cellular process from happening. Ex. a decoy ncRNA may bind to a microRNA (miRNA). The function of miRNA is to inhibit the translation of a particular mRNA. The decoy acts as a sponge by binding to the miRNA preventing function

Abnormalities in ncRNA

associated w/ a wide range of human diseases, we're still at the tip of the iceberg w/ regards to identifying the roles of ncRNA in human pathology

Crispr acronym

clustered, regularly interspaced, short, palindromic repeats

miR156, miR157, & miR159

control the time of year when flowering occurs

Telomeres

ends of eukaryotic chromosomes, a short nucleotide sequence that is repeated a few dozen to several hundred times in a row. 5'-GGGTTA-3' is the sequence found in human telomeres (flowering plants: 5'-GGGTTTA_3'). A telomere has a region at the 3' end termed a 3' overhang, bc it doesn't have a complementary strand.

Telomerase

enzymes that prevents chromosome shortening by attaching many copies of a DNA repeat sequence to the 3' ends of chromosomes. Telomerase contains both proteins & an ncRNA called telomerase RNA component (TERC). The lengthening of telomeres occurs in 3 steps.

Small nucleolar RNAs (snoRNAs)

found in high amounts in the nucleolus, & play a role in the covalent modification of rRNAs: the methylation of ribose on the 2' hydroxyl group & the conversion of uracil to pseudouracil. These modifications are common in rRNAs, but not in other types of RNAs. The location of peptidyl transferase (catalyzes peptide bonds during translation). The functional role of these modifications isn't entirely understood, but they fine-tune the rRNA structure for optimization.

Histone Modification

histone modification may affect gene transcription. The modifications facilitated by HOTAIR are known to inhibit transcription. This inhibition can happen in 2 ways: 1. The modifications may directly inhibit the ability of RNA polymerase to transcribe the target gene. These histone modifications may prevent RNA polymerase from forming a pre-initiation complex. 2. Rather than directly affecting transcription, the histone modifications may attract other chromatin-modifying enzymes to the target gene, which would lead to further changes in chromatin structure that inhibit transcription

CRISPR-Cas System

like the immune system of bacteria & archea, it provides defense against foreign invaders. CRISPR-Cas systems are an effective defense against bacteriophages (viruses that infect bacteria), & transpons (small segments of DNA that can be inserted into the chromosomes of all species). ncRNAs play a key role in the CRISPR-Cas system. About half of all bacterial species & most archeal species have such a system. 3 general types are known, type I, II, & III. (Also called CRISPR locus)

COOLAIR

ncRNA that promotes vernalization, the process in which certain plants will only flower

Small-Interfering RNAs (siRNAs)

ncRNAs that usually originate from sources that are exogenous, meaning they aren't normally made by cells. siRNAs can come from viruses that infect a cell, or it is made to be studied experimentally. In most cases, siRNAs are a perfect match to a single type of mRNA.They're thought to play a key role in preventing certain viral infections. siRNAs have become an important experimental tool in molecular biology.

MicroRNAs (miRNAs)

ncRNAs transcribed from endogenous eukaryotic genes- genes normally found in the genome. They play key roles in regulating gene expression, particularly during embryonic development in plants & animals. Most commonly, a single type of miRNA inhibits the translation of several different mRNAs. An miRNA & mRNA bind to each other bc they have sequences partially complementary. In humans, over 2,000 genes encode miRNAs. Researchers estimate that 60% of human protein-encoding genes are regulated by miRNAs.

CRISPR Adaptation (spacer acquisition)

occurs after a bacterial cell has been exposed to a bacteriophage. The proteins encoded by the Cas 1 & Cas 2 genes form a complex that recognizes the bacteriophage DNA as being foreign & cleaves it into small pieces. A piece of bacteriophage, usually between 20 & 50 bp in length, is inserted into the Crispr gene. The mechanism of insertion isn't entirely understood. The newly inserted piece of bacteriophage DNA is called a spacer bc it acts as a space between adjacent repeats. The different spacers found in the Crispr gene of modern bacterial species are derived from past bacteriophage infections. Each spacer provides a bacterium w/ defense against a particular bacteriophage. Once a bacterial cell has become adapted to a particular bacteriophage, it will pass this trait on to its daughter cells. By cleaving the bacteriophage into pieces, the adaptation phase can protect a bacterial cell, bc it cuts up the bacteriophage DNA & thereby inactivates the phage. However, a more effective way of destroying phages is provided by the expression & interference phases of this system.

RNA interference (RNAi)

phenomenon in which double-stranded RNA caused the silencing of mRNA. Found in most eukaryotic species, including animals & plants. The RNA that promotes RNA interference can come from 2 sources: microRNAs & small-interfering RNAs.

ncRNA binding

some ncRNAs bind to DNA or another RNA through complementary base pairing. This allows ncRNAs to affect processes such as DNA replication, transcription, & translation. ncRNAs can also bind to proteins or small molecules. Stem-loop structures (tRNA) may bind to pockets on the surface of proteins, or multiple stem-loops may form a binding site for a small molecule. In some cases, a single ncRNA may contain multiple binding sites. This allows it to facilitate the formation of a large structure composed of multiple molecules. (ncRNA & 3 different proteins)

ncRNA scaffolding

some ncRNAs contain binding sites for multiple components, such as a group of different proteins. An ncRNA can act as a scaffold for the formation of a complex. (stem-loop)

ncRNA can effect

the ability of mRNAs to be translated or degraded

Fire & Mello Experiment

the goal was to understand how the experimental injection of RNA was responsible for the silencing of particular mRNA's. The mex-3 gene was studied by making sense & antisense strands of mex-3 RNA. Some C. elegans received injections of antisense RNA while others received a mix of sense & antisense RNA, which formed a double-stranded RNA. In the embryos that received antisense RNA, the mex-3 mRNA levels were decreased; in the embryos that recieved the double-stranded RNA, no mex-3 mRNA was detected -> double-stranded RNA caused mex-3 mRNA to be degraded, this was termed RNA interference (RNAi).

Researchers & CRISPR-Cas system

they modify certain components of the system & use the to mutate genes in living cells.

ER signal sequence

to be directed to the ER membrane, a polypeptide must contain a sorting signal called a ER signal sequence, which is a sequence of about 6-12 amino acids that are predominantly hydrophobic & usually located near the N-term. As the ribosome is making the polypeptide in the cytosol, the ER signal sequence emerges from the ribosome & is recognized by a protein in SRP. The binding of SRP to the polypeptide pauses translation, SRP then binds to an SRP receptor in the ER membrane, which docks the ribosome over a channel. For this binding to occur, proteins within SRP & SRP receptor must also be bound by GTP. Next, these GTP-binding proteins hydrolyze their GTP, which causes the release of SRP from the SRP receptor & polypeptide. Once SRP is released, translation resumes & the growing polypeptide is threaded through a channel to cross the ER membrane. In this case of a secreted protein, the newly made polypeptide then travels through the golgi apparatus & then to the plasma membrane, where it is released outside the cell.

CRISPR-Cas system genes

tracr, Cas 9, Cas 1, Cas 2, & Crispr