Bentzen 7 - RNA molecules and mRNA processing part 2

Question 1

What is a summary of transcription and translation in bacteria?

Answer 1

Transcription produces fully functional mRNA

The mRNA start and stop is determined by the Shine-Delgarno sequence (ribosome binding), start codon and stop codon.

Question 2

How are genes organized in bacteria?

Answer 2

In operons

Question 3

Is mRNA polycistronic or monocistronic in bacteria?

Answer 3

Polycistronic, meaning there are several open reading frames (ORF) in one mRNA (leading to multiple polypeptides)

Question 4

What are two theories for how introns arose?

Answer 4

1. Introns have been around forever, but were lost in prokaryotes
2. Introns arose from group II introns, which themselves arose as parasitic DNA sequences (transposable elements). Subsequently invaded eukaryotes from endosymbiont ancestors of mitochondria and chloroplasts

Question 5

What is alternative processing of pre-mRNA intorns? What are two ways to do this?

Answer 5

A single pre-mRNA can be processed in different ways to produce different mRNA molecules.

1. Alternative splicing: pre-mRNA can be spliced in different ways
2. Alternative PolyA site: PolyA tail can be added at different 3’ cleavage sites

Question 6

What are four pre-mRNA splicing patterns?

Answer 6

1. Exon skipped (an exon is spliced out with introns)
2. Intron retention (an intron is left in the mRNA)
3. Alternative 5’ or 3’ splice site (A splice site is moved, including some of a intron or excluding some of an exon)
4. Mutually exclusive exons (Some exons are included only when another is excluded)

Question 7

What is the DSCAM gene ans its protein?

Answer 7

A gene encoding the Down Syndrome Cell Adhesion Molecule. It has an essential role in establishments of neural circuits.
The protein is a trans-membrane protein presented on surface of neurons.

The gene is about 600 million years old and in Down Syndrome critical region of chromosome 21 in humans

Question 8

How does the DSCAM RNA transcript demonstrate alternative splicing in Drosophilia?

Answer 8

There are 115 exons but only 20 are always expressed, and only 24 exons used in the final protein. There are 4 variable clusters of exons (total of 95 exons which can be alternatively spliced), one exon used from each cluster.

This leads to different isoforms of the protein. These isoforms are thought to be important in self recognition of neurons, and therefore important for proper neural wiring. In vertebrates it is different, the gene is duplicated, but much simpler in organization.

Question 9

How do mRNAs produce similar proteins of differing sizes when translated by alternative Poly(A) 3’ cleavage site?

Answer 9

Pre mRNA contains multiple 3’ cleavage sites. The 3’ cleavage site used determines the length of the mRNA transcript.

Question 10

How do alternative 3’ splice sites lead to production of a functional or nonfunctional protein in Tra pre-mRNA?

Answer 10

Transformer protein (Tra) in Drosophilia can be spliced at one site to be functional (and make a female phenotype) or nonfunctional (and cause male to stay male) depending on where the pre-mRNA is spliced

In males there is a premature stop codon when the upstream 3’ splice site is used (causing the Tra protein to be nonfunctional)

Question 11

In female drosophilia, the presence of what protein causes the downstream 3’ splice site to be used instead of the upstream one (which would cause a male)?

Answer 11

The Sxl protein, this splicing causes the termination codon to be spliced out with the intron

Question 12

Why do thyroid cells make Calcitonin and brain cells make calcitonin-gene-related peptide (CGRP) from the same DNA sequence?

Answer 12

In pre-mRNA processing, there is alternative processing based on the cell type.

Question 13

What are two ways to edit the coding information of mRNA transcripts? (when a gene is found with a sequence of nucleotides that do not exactly match that of its RNA product)

Answer 13

1. Substitution editing

2. Insertion/deletion editing

Question 14

What is substitution editing of mRNA?

Answer 14

Chemical alteration of individual nucleotides by specific enzymes.

Question 15

What is insertion editing of mRNA?

Answer 15

Addition of U nucleotides occurs by cleavage of the RNA, addition of the U, and ligation of the ends.

The reactions are catalyzed by a complex of enzymes under the direction of guide RNA

Question 16

What does guide RNA do in insertion editing of mRNA?

Answer 16

Guide RNA adds nucleotides to the pre-mRNA that were not encoded by the DNA

Question 17

What are RNA interferences (RNAi)?

Answer 17

Short regulatory RNAs that repress or ‘silence’ the expression of homologous genes

Question 18

What do the short (21-25 nucleotides long) utilize RNA molecules, siRNA and miRNA do? What are the three mechanisms that allow them to do this?

Answer 18

siRNA (small interfering RNA) and miRNA (microRNA) inhibit the expressino of homologous genes via at least 3 mechanisms:

1. mRNA degradation
2. Inhibition of translation
3. Inhibition of transcription

Question 19

How was RNA interference (RNAi) discovered?

Answer 19

By observing RNA silencing, a gene incorporated into the genome (a transgene) could not only induce or stimulate gene activity but could also inhibit the expression of homologous sequences.

In a flower a extra copy of a pigment gene (transgenes) added by researchers made parts of the flower turn white, rather than have more colour.

Question 20

What does double stranded RNA do in RNAi?

Answer 20

dsRNA triggers suppression of a gene in a homology dependent manner

Question 21

What are siRNA and miRNA made from? How does this cause gene regulation?

Answer 21

1. Cleavage of double stranded RNA by the Dicer enzyme.
2. The siRNAs are then combined with protein complex RISC and pair with complementary sequences on mRNA
3. The RNA-induced silencing complex (RISC) is made, which cleaves the mRNA and causes it to degrade

Question 22

How is gene regulation by inhibition of translation done?

Answer 22

1. Other double stranded regions of RNA molecules are cleaved by Dicer
2. This produces microRNAs (miRNA)
3. Some miRNAs combine with protein complex RISC and pair imperfectly with an mRNA
4. Which leads to the inhibition of translation

Question 23

How is gene regulation by inhibition of transcription done? What is RITS?

Answer 23

1. Other miRNAs attach to complementary sequences in DNA and attract methylating enzyme (RITS is a miRNA that attaches to do this)
2. Methylating the DNA or histones inhibits the transcription

RITS = RNA transcriptional silencing

Question 24

What percent of genes are thought to be regulated by RNAi? When is RNAi most important?

Answer 24

30%. During development

Question 25

Why might RNAi have evolved?

Answer 25

As a defence mechanism for silencing invading transposable elements

Question 26

Identify four ways to vary the products of genes and how they are expressed:

Answer 26

Alternative splicing

Alternative 3’ cleavage & polyA sites

RNA editing

RNA interference