BMB508: Noncoding RNAs

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41 Terms

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What is a noncoding RNA?

Noncoding RNAs are functional RNA molecules that is not translated into proteins.

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What is the primary function of noncoding RNAs?

They regulate gene expression where they can regulate gene expression at the levels of transcription, posttranscription (RNA processing) and translation.

Example! They are used in RNA splicing, gene regulation, telomere maintenance, and many other processes

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Why does the number of protein-coding genes show a poor correlation with genome sizes and/or number of different cell types?

In short - the non-coding regions take up more "space" than the coding regions, and are therefore a better indicator of genome size and number of different cell types (overall complexity)

- Regulatory elements: non-coding DNA includes regulatory elements (promoters, enhancers, and TF binding sites).

- The more complex an organism, the more sophisticated regulation is required (i.e. more regulatory sequences, hence more non-coding DNA)

- Splicing: non-coding regions contain alternative splice sites

Processes such as splicing, miRNA regulation and RNA editing generate a high proteome complexity from finite amounts of genetic information.

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What is a ribozyme and what does it do?

It is ribosomal RNA (noncoding RNA) and it catalyzes peptide bond formation

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What is a RNAse P?

Ribonuclease P is a ribozyme. It is a type of ribonuclease which cleaves RNA. Its function is to cleave off an extra, or precursor, sequence of RNA on tRNA molecules

It cleaves at 5' of tRNA.

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Why is the structure conserved and not the nucleotide sequences of the noncoding RNAs? What does this mean?

The function of the RNA lies in the structure. By conserving the structure, the function of the RNA is still valid.

That's why evolution favors RNA structure compared to the sequences.

- Lacking species conservation does not imply lack of function!

- Noncoding RNA might have species-specific function (eg. mouse but not human cardiomyocyte development, XCI, regulation of DNA methylation).

- NcRNA function encoded by ncRNA structure / function, not primary sequence.

- NcRNA conserved by structure, not primary sequence.

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What are circular RNAs (CircRNAs)?

Circular RNAs (CircRNAs) are single-stranded, covalently closed RNA molecules that are ubiquitous across species ranging from viruses to mammals.

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How are circular RNAs generated (CircRNAs)?

They are generated from 5'-3' backwards splicing

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What are the functions of circular RNA (CircRNAs)?

CircRNAs exert complex types of gene regulation such as:

- Decoys for small RNAs

- Binding of RNA-binding proteins

- Recruitment splicing factors

- Translation

- Exonuclease activity

- Biomarker

- Recombinant protein production

These functions are caused by the stability of circular RNAs so they can act as "sponges" for other small RNAs such as miRNA, RNA-binding proteins, and splicing factors.

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What features make circular RNA different from linear spliced mRNA?

CircRNAs does not produce classical cDNAs as they "can" contain introns and exons.

Nonetheless, their protein-coding potential is low, and few circRNA-derived peptides have been shown. One prominent feature is their stability, which makes them excellent 'sponges' for miRNAs, RNA-binding proteins, and splicing factors.

Their resistance is due to the lack of ends which makes them bad substrates against exonuclases like RNAse P.

Another difference is the lack of mRNA processing such as the addition of 5'Caps and poly-adenylation.

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Why can telomers be used as a cellular timestamp?

A cell is limited to how many times they can divide becomes of the telomere. The telomere protects the ends of the chromosomes but due to a insufficient telomerase expression, telomeres shorten gradually with each cell division in human somatic cells, which limits the number of times they can divide.

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What is TERC RNA?

Telomerase RNA component, also known as TR, TER or TERC, is an ncRNA found in eukaryotes that is a component of telomerase, the enzyme used to extend telomeres.

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What is the function of TERC RNA?

Provides template for telomeric DNA synthesis and scaffolds protein assembly

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How are telomers extended?

They are extended by the enzyme telomerase that adds the sequence "TTAGGG"

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What do telomerase need for it's activty?

It needs noncoding TERC RNA for it's telomerase activity because:

- It doesn't need a DNA template BUT!

- It requires RNA-DNA based pairing (RNA template) which can be brought by TERC RNA

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What are micro-RNAs?

MicroRNAs are small Noncoding RNAs that act as post-transcriptional regulators of protein abundances when present in their mature form

- In animal cells, miRNAs regulate their targets by translational inhibition and mRNA destabilization.

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What is the function of miRNAs?

Regulation of gene expression

- They can bind to a mRNA and silence it => No protein

- Their function is tightly correlated to the abundance of miRNA.

- They act as a buffer to protein levels, so we only get the optimal protein out (theostat)

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What was the first miRNA-mRNA network?

The lin-14/lin-4 where

- lin-4 is the miRNA that would be able to decrease the level of mRNA lin-14.

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What are the steps in canonical miRNA processing and gene regulation?

(1) pre-miRNA transcription

(2) pre-miRNA trimming

(3) Nuclear Export

(3/4) Dicing

(4/5) Repression/Degradation

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(1) What happens in the pre-miRNA transcription?

Primary miRNA or pri-miRNAs are transcribed by Polymerase II or III in the nuclear compartment in a process called pre-miRNA transcription.

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(2) What happens in the pre-miRNA trimming?

Pri-miRNAs are processed into precursor miRNAs or pre-miRNAs in the nucleus by the microprocessor complex (Drosha, Dgcr8) in a process called pri-miRNA trimming.

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(3) What happens in the nuclear export of the pre-miRNAs?

Pre-miRNAs are translocated into the cytoplasm by

Exportin (Xpo) proteins in a process called nuclear export.

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(3/4) What happens with the dicing of pre-miRNAs?

Pre-miRNAs are processed in the cytoplasm into a complex

of mature miRNAs (duplex) by endonuclease Dicer1 in a process called Dicing.

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(4/5) What is the repression/degradation step about when it comes to miRNA?

One mature miRNA strand is incorporated into the RNA interference silencing complex (RISC) in the cytoplasm, recognized by Argonaute proteins and bound to target mRNAs, which are subsequently de-adenylated or degraded aka slice or silence a mRNA, so it doesn't become a protein!

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How does miRNA target mRNAs?

They target with a 3' UTR binding

miRNAs bind to the 3′-untranslated region (3′-UTR) of target mRNAs and induce mRNA deadenylation and decapping

- The silencing depends on the nature of the miRNA elements and the miRNA abundance.

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Why is abundance important for the miRNA's function?

The function of miRNAs closely correlates with their abundance in cells.

- Highly expressed miRNAs have more chances to find and bind their targets and higher potency to repress their targets.

- The availability and abundancy of miRNAs and their target mRNAs are also contributing factors in determining which genes are regulated

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How can miRNA help in endocrine regulation?

The microRNAs (miRNAs) can be secreated into circulation since they are stable in plasma.

- They are then transported by lipoproteins/vesicles until it reaches the liver.

- The serum miRNA can then inhibited the genes that are expressed in the liver.

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What is antisense therapy and why is it related to miRNAs?

Antisense therapy aims to modulate the activity of specific miRNAs that are involved in disease processes by using synthetic molecules to inhibit their function

- By targeting miRNAs, antisense therapy offers a way to restore normal gene expression patterns and potentially treat various diseases. By targeting miRNAs, antisense therapy offers a way to restore normal gene expression patterns and potentially treat various diseases.

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Why would we target miRNAs for antisense therapy?

In many diseases, certain miRNAs become overactive or underactive, leading to dysregulation of

gene expression and contributing to the disease progression.

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Why would we inhibit miRNA for antisense therapy?

By inhibiting the miRNA from binding to its target mRNA, we prevent the miRNA from suppressing the expression on specific genes.

So, by targeting miRNAs, antisense therapy offers a way to restore normal gene expression patterns.

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What are the therapeutic advantages of using miRNAs?

miRNA have several target genes which partially fall into signaling pathways and functionally interrelated gene sets.

Targeting a single miRNA allows to de-repress sets of genes, in contrast to therapies that only target one mRNA through antisense oligonucleotides (ASO) and/or siRNAs.

Further, targeting miRNAs allows to increase protein abundances of their targets, which is challenging in the field of therapeutic oligonucleotides.

- Introduction of mRNA using liposomes and/or viral vectors might change this though in the foreseeable future

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What are long coding RNAs (lncRNAs)?

Long non-coding (lnc) RNAs are non-coding RNAs longer than 200 nt.

- lncRNAs primarily interact with mRNA, DNA, protein, and miRNA and consequently regulate gene expression at the epigenetic, transcriptional, post-transcriptional, translational, and post-translational levels in a variety of ways.

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What features are the shared between lncRNAs and mRNAs?

1) Common key epigenetic marks like promoter associated H3K4me3 or H3K36me3.

2) Expressed in specific cell-types and distinct developmental stages.

3) Both are poly-adenylated, albeit the length of polyA tails might differ.

4) Both are spliced, the splicing fidelity might be lower in lncRNAs.

5) Both typically are of a nucleotide length > 200 nucleotides.

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What features do lncRNAs have that are different to mRNAs?

1) Coding mRNAs typically have longer gene structures and thus higher number of exons. lncRNAs are of a more simplistic gene structure of 1-2 exons.

2) lncRNAs have a lower coding potential, thus encode no proteins and/or only short peptides or micropeptides.

3) lncRNAs are often found in the nucleoplasm, chromatin-associated or in specific cellular compartments, whereas spliced mRNAs are in the cytoplasm.

4) lncRNAs genomic sequences are less conserved between species.

5) lncRNAs can have -cis or -trans regulatory functions in gene regulation, mRNAs only the latter.

6) lncRNAs fold into complex 3D structures that can interact with nucleic acids and proteins, mRNAs less so.

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Long noncoding RNAs function when regulating in -cis?

cis -acting lncRNAs function at the site of transcription and affect the expression of neighbouring genes.

1) Sequence-specific functions

2) Transcription or splicing

3) DNA elements within the lncRNA

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Long noncoding RNAs functions when regulating in -trans?

Trans -acting lncRNAs function away from the site of synthesis.

1) Interactions with promoters and/or enhancers

2) Components of dynamic subnuclear structures or interchromosomal interactions

3) Regulation of RNA/protein activity in a stoichiometric manner

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Examples of long noncoding RNA functions!

HOTAIR recruits chromatin repressors

• HOTTIP opens chromatin by SET1-MLL recruitment

• HOTAIR activates transcription

• MALAT1 controls splicing

• BACE1AS increases BACE1 (miRNAs competition)

• PRNCR1 inhibits the androgen receptor TF

• GAS5 acts as decoy to glucocorticoid receptor

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What is another thing that lncRNAs can do?

lncRNA controls complex ncRNA circuits

Regulatory network:

2 miRNAs

1 lncRNA

1 circRNA

- lncRNAs Cyrano cleaves miR-7

- miR-7 loss increases target mRNAs

- miR-7 degrades circRNA Cdr1as

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What are ceRNAs?

ceRNAs: Crosstalk of lncRNAs / mRNAs with similar microRNA binding elements (MRE).

- The ceRNA compete for binding of miRNA thus higher translation of the mRNA (eg. PTENP1, L6-1 s.21).

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What are piRNAs?

The primary functions of piwi-interacted RNAs are silencing transposable elements!

RNA that directs the modification of chromatin to repress transcription; best characterized in the male germline where it inhibits transcription of transposons and is essential for spermatogenesis.

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What are siRNAs?

Primary function: inhibit translation by degrading mRNA.

Small interfering RNA's that form perfect duplexes with target mRNAs and cause mRNA cleavage by the RNA-mediated interference pathway.