CH 17 - Non-Coding RNAs

Coding RNAs

• generally refers to mRNA that encodes protein

Noncoding RNAs (ncRNAs)

• act as cellular regulators w/o encoding proteins

• carry out many functions because they can bind to different types of molec

  • DNA: via complementary base pairing

  • Other RNAs: via complementary base pairing

  • Proteins

• ncRNA molec can form stem-loop structures, which may bind to pockets on the surface of proteins

ncRNAs functions

• scaffold: ncRNA binds to group of proteins

• guide: binds to a protein and guides it to a specific cite in cell

• alt of protein function/stability: binds to a protein and alters that protein’s structure

• ribozyme: RNA molec w/ catalytic function

• blocker: physically prevents/blocks a cellular process from happening

• decoy: recognizes another ncRNA and isolates it

Long Non-coding RNAs (lncRNAs)

• longer than 200 nucleotides

  • microRNA (miRNA)

  • small nuclear RNA (snRNA)

  • small nucleolar RNAs (snoRNAs)

  • short interfering RNAs (siRNA)

  • PIWI-interacting RNAs (piRNAs)

Small non-coding/reg RNAs (aka ncRNAs)

• shorter than 200 nucleotides

ncRNA effects on chromatin structure and transcription

• Hox transcript antisense intergenic RNA (HOTAIR) - recently discovered ncRNA that alters chromatin structure

• the HOTAIR gene is located on human chromosome 12 within a cluster of genes called the HoxC genes

• 2.2-kb-long ncRNA

• transcribed from the opp (antisense) strand w/ respect to the HoxC genes

• acts as scaffold that guides two histone-modifying complexes to target genes

Mechanism of HOTAIR Transcriptional Repression

• acts as scaffold for the binding of 2 protein complexes (PRC2 & LSD1)

  • Polycomb Repressive Complex 2

  • Lysine specific Demethylase (LSD1)

• Binds PRC2 to the 5’ end and LSD 1 to the 3’ end

• Guides the 2 protein complexes to a GA-rich region

• GA-rich region contains many purines

• PRC2 functions as a histone transferase

  • Trimethylates lysine 27 on histone H3

• LSD1 demthylates mono- and demethylated lysines

ncRNAs: effects on translation and mRNA degradation

• ncRNAs can affect the ability of mRNAs to be translated or degraded

Example of RNA silencing in petunias

• researchers Jorgensen and Stuitje attempted to produce strains of petunias w/ deeper flower colors

• methodology: involved inserting into petunia genome copies of cloned genes that coded enzymes involved in the synthesis of flower pigment

• findings: flower pigmentation in some cases did not deepen, but instead showed variegation

• conclusions: additional copies of gene can sometimes suppress the expression of both itself and its endogenous counterpart

  • later research found the production of double-stranded RNA to be involved in lowering mRNA levels

Injection of antisense and double-stranded RNAs into C. elegans to compare their effects on mRNA silencing

• Involved in mRNA coded by a gene, mex-3 (Andrew Fire and Craig Mell)

  • abundant mRNA in early embryos of C. elegans

  • sense and antisense mex-3 RNA were made in vitro using cloned genes for mex-3 w/ promoters on either side of gene

  • RNA pol and nucleotides added to synthesize RNA

  • either mex-3 antisense RNA or a mix of mex-3 sense and antisense RNA injected into the gonads of C. elegans

  • RNA is taken up by the eggs and early embryos, control, not injected w/ any RNA

  • incubation and in situ hybrid of early embryo

  • a labeled probe is added that is complementary to mex-3 mRNA

    • if cells express mex-3, the mRNA in the cells will bind to the probe and become labeled

  • after incubation w/ a labeled probe, cells were washed to remove unbound probe

  • embryos were observed under the microscope

Andrew Fire and Craig Mello: Interpreting the Data

• the data indicate that double-stranded RNA is more potent at silencing mRNA than is antisense RNA

• mex-3 mRNA was completely degraded

• RNA interference (RNAi): describes phenomenon of double-stranded RNA causes silencing of mRNA

RNA interference is mediated by microRNA/small-interfering RNAs

• RNA interference is found in most euk species

• mediated by 2 types of ncRNAs: microRNA (miRNA) & small-interfering RNAs (siRNA)

MicroRNAs (miRNAs)

• transcribed from endogenous euk genes

• reg gene expression

  • single type of miRNA inhibits the translation of several diff mRNAs thru partial complemntarity

  • 60% of human genes may be reg by microRNAs

Small interfering RNAs (siRNAs)

• ncRNAs that usually originate from exogenous sources (not normally made by cells)

• can be from viruses/experimentally injected by researchers

Mechanism of RNA interference

• 1st pri-miRNA is made

• makes hairpin recog by 2 proteins, Drosha and DGCR8

• Cleaved @ both ends into 70 nucleo pre-miRNA and exported from nucleus

• cut by dicer to 50-25 bp

• short souble-stranded RNA assoc w/ proteins to form RNA-induced silencing complex (RISC)

• 1 RNA strand degrades, the other is complementary to the mRNA that is silenced

• siRNA don’t go thru processing events that occur in nucleus (they’re either derived from viral RNAs or introduced into cell by researchers)

RNA-induced silencing complex (RISC)

• after RISC binds to an mRNA, the effects may be:

  • RISC may inhibit translation w/o degrading the mRNA, often partially complementary to their mRNAs

  • processing body (p-body) stores RISC mRNA until it is used/degraded

  • RISC may direct degradation of MRNA thru cleavage by Argonaute (occurs w/ siRNAs)

Functions and benefits of RNA interference

• RNAi is vital for gene reg in plants and animals

  • production of miRNAs silences the expression of specific mRNAs

• RNAi provides defense against viruses

• siRNA can inhibit transc by causing chromatin modifications

  • effect shared w/ piRNAs

Non-coding RNAs: Effects of RNA modifications

• small nucleolar RNAs (snoRNAs) are found in high amounts in nucleolus

• synthesis of ribosomal RNAs (rRNAs) and assembly of ribo subunits occurs in nucleolus

• snoRNAs covalently modify RNAs

  • methylation of ribose on the 2’ hydroxyl group

  • conversion of uracil to pseudouracil

C/D box snoRNAs

• 70-120 nucleotides and guide methylation of target RNAs

• form snoRNP w/ 2 copies of a protein that catalyzes the methylation of ribose

H/ACA box snoRNAs

• ~100-200 nt and guide pseudouridylation

• forms a snoRNP w/ two copies of a protein that catalyzes the conversion of uracil to pseudouracil

• small nucleolar ribonucleoprotein: ea type provides a scaffold to form ““

snoRNAs also act as guides

• snoRNAs have antisense seq that are complementary to sites in RNAs

• guide the proteins in the snoRNP complex to theirtarget rRNAs for chemical modification

  • methylation of ribose

  • conversion of uracil to pseudouracil

Effects if ncRNAs are expressed abnormally

• disease conditions occur

  • cancer

  • multiple sclerosis

  • alzheimer’s disease

  • heart arrhythmias/heart fail