RNA in Cell Signalling - Week 1

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Questions and Answers

What is the primary role of ribosomal RNA (rRNA) in the cell?

  • Major structural component of the ribosome (correct)
  • Guiding chemical modification of RNA
  • Translating mRNA signals into proteins
  • Interacting with small nuclear RNA

Less than 2% of the RNA transcribed from the human genome is non-coding.

True (A)

What class of non-coding RNA is involved in the modification of pre-mRNA?

Small nuclear RNA (snRNA)

The largest class of small non-coding RNA is __________.

<p>Piwi interacting RNA (piRNA)</p> Signup and view all the answers

Match the non-coding RNA with its function:

<p>Ribosomal RNA (rRNA) = Major structural component of ribosome Transfer RNA (tRNA) = Involved in translation of mRNA Small nuclear RNA (snRNA) = Pre-mRNA modification Small Nucleolar RNA (snoRNA) = Guides chemical modification of RNA</p> Signup and view all the answers

What percentage of the human genome is transcribed into RNA?

<p>75% (C)</p> Signup and view all the answers

Transfer RNA (tRNA) is solely responsible for coding proteins.

<p>False (B)</p> Signup and view all the answers

What are the two classes of small nuclear RNA?

<p>smRNA and lsmRNA</p> Signup and view all the answers

What type of gene knockdown is typically associated with shRNA transfection?

<p>Stable gene knockdown (B)</p> Signup and view all the answers

SiRNA is typically used for long-term gene expression changes.

<p>False (B)</p> Signup and view all the answers

What is a key requirement for a successful overexpression system in higher eukaryotes?

<p>A suitable cell line and a suitable vector</p> Signup and view all the answers

Adenovirus vectors are well suited for _____ due to their ability to transduce both dividing and non-dividing cells.

<p>oncolytic therapy</p> Signup and view all the answers

Match the following vector types with their descriptions:

<p>Adenovirus vectors = Carry up to 8.5 Kbs of heterologous DNA Retroviral vectors = RNA viruses that replicate via a dsDNA intermediate Vaccinia vectors = Replicates in the cytoplasm of the host cell Plasmid-based expression vectors = Common tool for gene cloning and expression</p> Signup and view all the answers

What is the primary function of short interfering RNA (siRNA)?

<p>Cleaving RNAs derived from viruses and repeat sequences (A)</p> Signup and view all the answers

Micro RNA (microRNA) is involved in translational repression or cleavage of target mRNAs.

<p>True (A)</p> Signup and view all the answers

What is the purpose of providing a homologous repair template in gene editing?

<p>To facilitate precise mutations and knock-ins (A)</p> Signup and view all the answers

Indel refers to the combination of insertion and deletion of bases in an organism's genome.

<p>True (A)</p> Signup and view all the answers

What are the most common group of non-coding RNAs (ncRNAs) that are over 200 bases in length?

<p>Long non-coding RNA (LncRNA)</p> Signup and view all the answers

LncRNAs can act as either ___________ or ___________ transcriptional regulatory elements.

<p>cis, trans</p> Signup and view all the answers

What are the two main mechanisms of RNA interference?

<p>Short interfering RNA (siRNA) and short hairpin RNA (shRNA)</p> Signup and view all the answers

CRISPR/Cas9 technology relies on __________ to create site-specific genomic modifications.

<p>site-specific nucleases</p> Signup and view all the answers

Match the following non-coding RNAs with their primary functions:

<p>siRNA = Cleaving RNAs from viruses microRNA = Translational repression of target mRNAs exRNA = Cell-cell communication scaRNA = Regulates spliceosome RNAs</p> Signup and view all the answers

Match the following terms to their definitions:

<p>Gene knockdown = A technique that reduces gene expression significantly RNA interference = Method involving the degradation of target mRNA Homology directed repair = Process that uses a repair template for precise modifications Nuclease-deficient Cas9 = Cas9 variant fused with effector domains for specific localization</p> Signup and view all the answers

Which long non-coding RNA is associated with the deactivation of X chromosome function?

<p>Xist (B)</p> Signup and view all the answers

Loss of regulation of LncRNAs has not been linked to human cancers.

<p>False (B)</p> Signup and view all the answers

Which type of RNA is produced during RNA interference?

<p>Anti-sense RNA (D)</p> Signup and view all the answers

Nuclease-deficient Cas9 is used to introduce double-stranded breaks in DNA.

<p>False (B)</p> Signup and view all the answers

What example of LncRNA has been noted for its role in cancer metastasis?

<p>HOTAIR</p> Signup and view all the answers

Extracellular RNA (exRNA) is believed to be involved in ___________ and ___________ regulation.

<p>cell-cell communication, cell</p> Signup and view all the answers

What is the result of using two sgRNAs with mutated Cas9?

<p>It introduces a staggered double-stranded break.</p> Signup and view all the answers

The formation of a __________ is crucial for the degradation of target mRNA during RNA interference.

<p>RNA induced silencing complex (RISC)</p> Signup and view all the answers

What role do LncRNAs play in relation to oncogenes and tumor suppressor genes?

<p>They can function as both oncogenes and tumor suppressor genes (A)</p> Signup and view all the answers

What is a potential outcome of gene knockdown?

<p>Temporary reduction of gene product levels (C)</p> Signup and view all the answers

Which technique involves the permanent deactivation of a specific gene within an organism?

<p>Gene knockout (D)</p> Signup and view all the answers

Homologous recombination does not utilize the cell's own DNA repair mechanisms.

<p>False (B)</p> Signup and view all the answers

What is one common method used to introduce synthetic DNA sequences into cells?

<p>Electroporation</p> Signup and view all the answers

As of 2009, approximately ______ genes had been knocked out of mice.

<p>11,000</p> Signup and view all the answers

Which of the following techniques is used to create engineered mutations directed to a specific locus?

<p>Homologous recombination (A)</p> Signup and view all the answers

Site-specific nucleases can be used to achieve gene knockouts.

<p>True (A)</p> Signup and view all the answers

What is the main purpose of gene knockdowns in research?

<p>To study gene function and its role in signaling pathways.</p> Signup and view all the answers

_______ is a technology that can be used in homologous recombination to target specific genes.

<p>CRISPR/Cas9</p> Signup and view all the answers

Match the following gene delivery techniques with their descriptions:

<p>Electroporation = Using an electrical field to increase cell membrane permeability Microinjection = Directly injecting DNA into a cell Viral vectors = Using viruses to deliver genetic material into cells Liposomes = Encapsulating DNA in lipid-based carriers</p> Signup and view all the answers

What type of RNA accounts for a large proportion of transcribed RNA within eukaryotic cells?

<p>Non-coding RNA (A)</p> Signup and view all the answers

Flashcards

Ribosomal RNA (rRNA)

A type of RNA that forms the core of ribosomes, the cellular machinery responsible for protein synthesis. rRNA interacts with mRNA during translation.

Transfer RNA (tRNA)

RNA molecules that transport specific amino acids to the ribosome during translation, ensuring the correct order of amino acids in the growing protein chain.

Small Nuclear RNA (snRNA)

Non-coding RNA molecules involved in various processes within the cell, including mRNA processing and splicing.

Small Nucleolar RNA (snoRNA)

Non-coding RNA molecules involved in modifying other RNA molecules, primarily rRNA.

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Piwi-interacting RNA (piRNA)

The largest class of small non-coding RNAs, playing a crucial role in silencing gene expression in germ cells.

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Short Interfering RNA (siRNA)

Small interfering RNA molecules that silence genes by targeting specific mRNA sequences for degradation, playing a key role in defense against viruses and repetitive sequences.

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Micro RNA (microRNA)

Small non-coding RNA molecules that regulate gene expression by binding to specific mRNA sequences, leading to either mRNA degradation or translational repression.

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Extracellular RNA (exRNA)

RNA molecules that exist outside of cells, participating in cell-to-cell communication and various regulatory processes.

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Small Cajal body-specific RNA (scaRNA)

Small non-coding RNA molecules found specifically in Cajal bodies, nuclear structures involved in RNA polymerase II transcription and splicing.

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Long Non-Coding RNA (lncRNA)

Non-coding RNA molecules longer than 200 bases, involved in regulating gene expression.

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Gene Knockdown

The process of reducing gene expression, leading to a decrease in protein levels.

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RNA Interference

A technique that uses small RNA molecules (siRNA or shRNA) to silence gene expression by degrading specific mRNA transcripts.

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CRISPR/Cas9

A powerful gene editing technology that allows precise modifications to the genome by introducing nicks or fusions at specific locations.

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shRNA

A type of RNA interference (RNAi) that utilizes small hairpin RNAs (shRNAs) to stably knock down gene expression for extended periods.

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siRNA

A type of RNA interference (RNAi) that utilizes small interfering RNAs (siRNAs) for transient gene knockdown, typically affecting gene expression for a shorter time.

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Overexpression

A technique used to increase the production of a specific protein in cells, typically by introducing a gene that encodes the desired protein.

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Adenovirus Vector

A viral vector used in gene delivery, known for its ability to express high levels of transgenes in both dividing and non-dividing cells.

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Retroviral Vector

A viral vector used in gene delivery, capable of integrating its genetic material into the host cell genome, leading to long-term expression of the transgene.

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Vaccinia Vector

A viral vector derived from the vaccinia virus, a double-stranded DNA virus that replicates in the host cell's cytoplasm.

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Plasmid-based Vector

A circular DNA molecule often used in recombinant DNA technology to carry and express genes of interest.

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Gene Knockout

A permanent gene deactivation technique that involves altering the target gene's DNA sequence to disrupt its function.

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Homologous Recombination

A gene knockout technique that utilizes homologous recombination, a process where DNA segments with similar sequences exchange.

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Site-specific Nucleases

A type of gene knockout technique that involves using enzymes like CRISPR/Cas9 to make precise cuts in the target gene's DNA sequence.

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Gene Expression Profiling

Analyzing gene expression patterns through methods like microarrays and RNA sequencing to understand cellular processes.

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Overexpression and Gene Knockout/Knockdown

Techniques like RNA interference (RNAi) and knockout approaches are crucial for studying the roles of genes in signaling pathways.

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Study Notes

RNA Overview

  • RNA plays a crucial role in the central dogma of molecular biology.
  • Over 75% of the human genome is transcribed to RNA, but less than 2% encodes proteins.
  • The remaining RNA consists of non-coding transcripts.

Non-Coding RNA (ncRNA) Classification

  • Ribosomal RNA (rRNA): Major component of ribosomes; interacts with mRNA.
  • Transfer RNA (tRNA): Facilitates translation of mRNA into proteins.
  • Small Nuclear RNA (snRNA): Involved in pre-mRNA modification; includes smRNA and lsmRNA.
  • Small Nucleolar RNA (snoRNA): Guides modifications of other RNA molecules.
  • Piwi-interacting RNA (piRNA): Largest class of small ncRNA; involved in gene silencing in germlines.
  • Short Interfering RNA (siRNA): Silences genes by degrading RNAs from viruses and repetitive sequences.
  • Micro RNA (microRNA): Regulates gene silencing through mRNA cleavage.
  • Extracellular RNA (exRNA): Participates in cell-cell communication and regulation.
  • Small Cajal body-specific RNA (scaRNA): Regulates RNA polymerase II activities within Cajal bodies.

Long Non-Coding RNA (LncRNA)

  • Defined as non-coding RNAs over 200 bases long, regulating gene expression and protein function.
  • Act as transcriptional regulatory elements, either enhancing or repressing transcription.
  • Example: Xist deactivates the X chromosome by interacting with polycomb-repressive complexes.
  • Changes in LncRNA regulation link to human cancers, with overexpression of HOTAIR associated with metastasis and tumorigenesis.

Gene Manipulation Techniques

  • Gene Knockdown: Alters gene expression, reducing protein levels significantly without complete deactivation; can be temporary or permanent.
  • RNA Interference: Transient gene knockdown via anti-sense RNA production, employing siRNA or shRNA mechanisms leading to mRNA degradation.
  • CRISPR/Cas9: Allows precise genome editing by introducing specific nicks or fusions for targeted gene modification.

Laboratory Applications

  • Investigating gene expression profiles helps understand cell signaling mechanisms.
  • Overexpression and gene knockout/knockdown methods are essential for studying signaling pathways.

Gene Knockout Techniques

  • Permanent gene deactivation with methods like homologous recombination and site-specific nucleases.
  • Homologous recombination has successfully knocked out approximately 11,000 genes in mice, equating to half the mouse genome.

shRNA vs siRNA

  • shRNA generally enables stable, long-term gene knockdown, while siRNA typically leads to transient gene silencing.

Overexpression Systems

  • Employed predominantly in mammalian cells using immortalized cell lines and suitable vectors to produce protein overexpression.
  • Common vector types:
    • Adenovirus Vectors: High transgene expression in dividing and non-dividing cells, carrying up to 8.5 kb of DNA.
    • Retroviral Vectors: RNA viruses replicating via a dsDNA intermediate, including lentiviruses.
    • Vaccinia Vectors: Double-stranded DNA viruses replicating in the host cell cytoplasm.
    • Plasmid-based Vectors: Common in recombinant DNA technology for protein expression.

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