BCMB TEST 1

Life of a Eukaryotic mRNA

*In nucleus*: DNA - Pol II transcription -> primary RNA transcript - nuclear processing (capping, methylation, polyadenylation, splicing, editing) -> mature mRNA -> in cytoplasm: mature mRNA - translation -> degradation into protein

three domains of life

Bacteria, Archaea, Eukarya

Argonaute family of proteins

Catalytic subunit of the RISC complex of the RNAi machinery. Responsible for the cutting (or "slicing") activity of RISC that destroys target mRNAs.

ADARs

Changes A>I, adenosine deaminases that act on RNA

Glutamate Receptor RNA Editing (A to I)

Example: Q/R site editing Adenosine is *deaminated* to inosine (CAG to CIG) Glu to Arg change alters calcium permeability of the channel which alters brain function

need for (mRNA) splicing

Introns are encoded in the primary RNA transcripts and must be removed by a process called splicing to generate functional RNA (i.e. mRNA, rRNA, tRNA, etc.)

metazoan

Multicellular animal

mRNA splicing

Process that removes introns from pre-mRNAs and joins exons together.

Why would Transcriptomes vary?

Stimuli yield responses (drugs, light, fear, chemicals, stress, etc.) Developmental stages require changes in gene expression Tissues need different proteins for function Disease states are associated with deviation from healthy state (e.g. cancer vs. normal cells)

The DNA of different cell types is generally __________ in both ___________ and _______ (notable exceptions do occur under rare circumstances). However, highly specialized cases are known to exist where DNA _____, ______________, and _______________ profoundly influence gene expression in isolated situations.

The DNA of different cell types is generally *similar* in both *amount and type*. However, highly specialized cases are known to exist where DNA *loss*, *rearrangement*, and *amplification* profoundly influence gene expression in isolated situations.

Transcriptomics vs. Proteomics

Transcriptomics and proteomics are both very powerful Differences in their practical application: Transcriptomics is robust, relatively cost-effective and user-friendly Proteomics still relatively limited -problems can remain with purification and stability of proteins

Macromolecular Interaction Analysis

Understanding protein/protein, protein/DNA and protein/RNA interactions in the cell 1. Co-immunoprecipitation or Co-selection Experiments 2. ChIP-Seq 3. CLIP: Crosslinking and Immunoprecipitation

RNA processing

a collection of events required to convert primary gene transcripts (precursor RNA) into biologically or functionally active RNA (mature RNA). All RNAs in all organisms are synthesized as primary transcripts, which must undergo RNA processing reactions; the major cellular RNAs (ribosomal RNA, transfer RNA, and messenger RNA) are formed by extensive processing of primary transcripts. 1. covalent events 2. noncovalent events

CRISPR/Cas System

a molecular tool used for precise editing of DNA that relies on the action of CRISPR RNAs and Cas proteins The discovery and application of CRISPR-Cas systems resulted in a Noble Prize in Chemistry in 2020 awarded to two truly amazing female scientists, Jennifer Doudna (Berkley) and Emmanuelle Charpentier (Berlin)

APOBEC

apolipoprotein B editing complex deaminase responsible for ApoB (C to U) editing

DNA Microarrays

hybridization-based approach Incubate fluorescently labelled cDNA (RNA copies)with DNA microarrays Intensity of fluorescent signal reflects abundancy of RNAs Used to estimate the relative levels of gene expression of each gene in a genome in a single experiment Primarily used to compare the expression of many genes under different conditions (e.g. cancer vs normal cells, treatment with drugs, etc.)

RNA world hypothesis

hypothesis that RNA served as the genetic information of early life questioned whether RNA was the primordial molecule from which life evolved ~ 3.8 billion years ago

miRNAs

micro RNAs

eukaryotic mRNA

monocistronic

prokaryotic mRNA

polycistronic

siRNAs

short interfering RNAs

steps of ChIP-Seq

*1. Treat intact cells or tissues with formaldehyde to induce covalent crosslinking between DNA and target DNA binding proteins. * *2. Lyse cells and sonicate to reduce the overall average size of RNAs to ~200-500 base-pair DNA fragments.* *3. Purify RNA-protein complexes by immunoprecipitation under stringent conditions using an antibody against the DNA binding protein (or an epitope tag if the protein was expressed as a fusion protein).* *4. Reverse the formaldehyde crosslinks by heating *5. Incubate the purified material with proteinase K (to destroy the proteins) leaving pure DNA.* *6. Perform high throughput DNA sequencing according to work flow using Illumina technology * *7. Map the DNA reads back to reference genome to identity the DNA region(s) that the protein was bound to in the living cell or tissue*

functional genomics

*Experimental-based approach to understand gene function (cellular phenotypes) on a genome-wide(or system-wide) scale* studies : Tissue-specific or temporal expression patterns Subcellular localization Physical interactions with other gene products or cellular components Genetic interactions with other gene products

Major pathway of mRNA degradation

1) Shortening of 3' poly(A) tail by a 3' to 5' exoribonuclease. 2) Decapping. 3) Degradation by 5' to 3' exoribonuclease

Pathway of Messenger RNA (mRNA) Synthesis

1) Synthesis by RNA Polymerase II in nucleus *(Birth)* 2) 5' capping 3) Internal base and sugar modification (e.g. 2'-O-me, m6A) 4) hnRNP assembly 5) 3' end formation/polyadenylation *(Maturation)* 6) Splicing (intron removal) 7) Editing (in rare, but significant cases) 8) Export (nuclear-cytoplasm transport) 8) mRNP protein binding 9) Translation or Degradation *(Fulfillment and Ultimate Death)*

types of changes in gene content or position

1. Gene Loss 2. Gene Amplification 3. Gene Rearrangement

steps of DNA Microarrays

1. Isolate mRNAs from cells at two stages of development; each mRNA sample represents all the gene expressed in the cells at that stage 2. convert mRNAs to cDNAs by reverse transcriptase, using fluorescently labeled deoxyribonucleotide triphosphates 3. add the cDNAs to a microarray; fluorescent cDNAs anneal to complementary sequences on the microarray 4. each fluorescent spot represents a gene expressed in the cells

Immuno or Affinity Purification of Protein Complexes

1. Purify via immunoprecipitation (if antibody available) or selection of 'tagged' protein Common protein 'tags'/affinity method: - epitope tags (myc, HA, FLAG) / antibodies - poly histidine (e.g. 6x) / nickel chromatography 2. Separate complexes (tagged protein and co-purifying proteins) on SDS gel 3. Excise individual protein bands 4. Digest with trypsin (cuts after Arg/Lys) 5. Identify proteins by mass spectrometry(produces charged particles (ions) and uses electric/magnetic fields to measure mass of peptides or direct peptide sequencing) 6. Data base analysis : Identify proteins from masses (or sequence) of peptides

what are the Four main levels of control in gene activity (expression patterns)? how do multiple control points change gene activity?

1. transcriptional regulation DNA -> primary RNA transcript 2.post-transcriptional regulation NUC: primary RNA transcript -> mRNA -> CYT: mRNA -> inactive mRNA or protein -> inactive protein 3. translational control 4. post- translational control

Size of human genome(bp)? % of human genome that codes for protein? % of human genome that is transcribed? % of total transcripts that are non-coding RNAs?

3.3 x 10^9 bp ~2% ~90% ~98%

Organization of a Typical Eukaryotic mRNA

5' cap - 5'UTR - AUG - coding region - UAA - 3'UTR - poly(A)tail

RNA Editing

A post-transcriptional alteration in the nucleotide sequence of an RNA Rare but functionally significant Editing can create two or more proteins from a single gene, remove frameshifts, create stop or start codons, or alter codons In mammals, only single nucleotide changes have been identified (base substitutions) In trypanosomes, mitochondrial mRNAs are extensively inserted / deleted. Small trans-acting guide RNAs provide the genetic info for these reactions.

A single ____ can give rise to two structurally/functionally different ____________ depending upon cell type (alternative splicing)

A single *gene* can give rise to two structurally/functionally different *proteins* depending upon cell type (alternative splicing)

Differentiation-specific gene expression

A single gene can give rise to more than one functionally distinct protein within the same cell. In some cases, the decision of alternative splice-site choice is regulated by the differentiation state of the cell

ChIP Seq Assays

ChIP = *Ch*romatin *I*mmuno*p*recipitation A method that combines chromatin immunoprecipitation and high throughput DNA sequencing to identify specific protein/DNA interactions in vivo and genome-wide -Transcription factors -Histones (various types of modifications) -RNA polymerase (investigate transcription globally) -DNA repair proteins etc. Method to detect if a particular protein is present at a particular genomic region in vivo Requires an antibody that recognizes the protein of interest

environmental gene expression

Changes in phenotype of an organism when genes are activated by a change in the environment, ex. Fur color/thickness in rabbits

Regulation of mRNA Stability

Chemical half-lives of mRNAs range from a few minutes to more than a day. The half-life of a particular mRNA (time it takes for 50%mRNA to be degraded once new synthesis is stopped) can be influenced by several factors including nutrient levels, hormones, viruses, and changes in temperature. Changing the rate of mRNA degradation usually changes the rate of protein synthesis.

Transcriptional control

Detemines if, how much, and when an mRNA is made. Control of transcriptional initiation is a primary means used to regulate gene expression in eukaryotic organisms. Genes are controlled at the level of transcription by proteins (trans-acting factors) that interact with specific gene sequences (cis-acting regulatory sequences)

Post-translational control

Determines if, how much, and when a protein is functional. Newly made proteins often undergo extensive chemical alterations in order to become functional. Examples include amino acid modifications (e.g. phosphorylation, acetylation, myristylation, methylation, and glycosylation) and proteolytic cleavages

translational control

Determines if, how much, and when a protein is made The amount of protein produced from a given mRNA can be regulated gene expression regulated by influencing the interaction of the mRNA transcripts with the ribosome

DsCAM

Drosophila homolog of human Down syndrome cell adhesion molecule an example of multiple proteins from one gene using alternative splicing 12 x 48 x 33 x 2 = 38,016 different proteins! (entire human genome encodes just ~20,000 proteins)

co-suppression

Expression of a transgene can paradoxically suppress (inactivate) expression of the corresponding endogenous gene. This effect was first identified in plants (the same phenomena identified in fungi was termed 'quelling')

tissue-specific gene expression

Genes are expressed in a specific manner to facilitate the function of different cell or tissue types. Ex. Leaf tissues in a plant making proteins for photosynthesis.

how do you map molecular interactions?

Identifying components of protein complexes: Immuno or Affinity Purification of Protein Complexes Identifying specific DNA/protein interactions: ChIP-Seq. Identifying specific RNA/protein interactions: CLIP-Seq. *Antibodies specific to the protein of interest are key reagents for these three techniques

In general, the information encoded in DNA is _____________ into RNA and ____________ into proteins.

In general, the information encoded in DNA is *transcribed* into RNA and *translated* into proteins.

occurrance of introns

Introns are present in all organisms (bacteria, achaea, and eukaryotes) in different organelles (mitochondria and chloroplasts) and even in some viruses. Introns are present in many different gene types (mRNAs, rRNAs (in some unicellular eukaryotes), tRNAs, snRNAs (in rare cases))

Level of "gene transcription" is ___________ to __________ of the fluorescence signal

Level of "gene transcription" is *proportional *to *intensity *of the fluorescence signal

Many eukaryotic genes contain multiple ________. Usually all ________ must be removed before the mRNA can be translated to produce protein.

Many eukaryotic genes contain multiple *introns*. Usually all *introns* must be removed before the mRNA can be translated to produce protein.

Crosslinking Immunoprecipitation (CLIP-Seq Assays)

Method to identify the RNAs that a particular RNA binding protein interacts with in vivo *UV irradiate cells (cross-link RNA and proteins) Partial RNase digestion (fragment RNAs)* *Purify RNP (Ip = immunoprecipitation)* *Proteinase K/DNase* *Ligate RNA linkers 5'+ 3'* *Convert RNA to DNA (RT-PCR)* *Sequence DNAs * *Database searching/Identification*

Eukaryotes vs Prokaryotes

Presence of a nucleus in eukaryotic cells. Multiple copies for many eukaryotic genes; large amount of non-coding DNA. Most eukaryotic genes are interrupted by non-coding DNA (e.g. introns), which is transcribed and later spliced out or otherwise removed. Coupled (proks) vs. uncoupled (euks) transcription / translation reactions Polycistronic (more than one protein/mRNA; common in proks) vs. monocistronic (one protein/mRNA) in euks. Extensive modification of eukaryotic mRNAs (e.g. 5' caps and 3' polyA tails, etc.). Multicellular (euk) vs. unicellular (prok) (differences in cellular environments/responses and expression regulation potential)

non-covalent events in RNA processing

RNA folding/unfolding. Protein binding (assembly into ribonucleoprotein complexes (RNPs)). Transport in cells

RISC and slicer

RNA induced silencing complex effector nuclease : Argonaute 2

ribozymes

RNA molecules that act as enzymes discovered by Sidney Altman and Thomas Czech, awarded the Nobel Prize in Chemistry in 1989

RNA-Seq vs. microarray

RNA-seq can be used to characterize novel transcripts and splicing variants as well as to profile the expression levels of known transcripts (but hybridization-based techniques are limited to detect transcripts corresponding to known genomic sequences) RNA-seq has higher resolution than whole genome tiling array analysis In principle, mRNA can achieve single-base resolution, where the resolution of microarray depends on the density of probes RNA-seq can apply the same experimental protocol to various purposes, whereas specialized arrays need to be designed in these cases Detecting single nucleotide polymorphisms (needs SNP array otherwise) Mapping exon junctions (needs junction array otherwise) Detecting gene fusions (needs gene fusion array otherwise

Two-dimensional Gel Electrophoresis

Separates proteins in two steps, first by isoelectric points (net charge) and then by molecular weights. proteins from E. coli separated by 2D-electrophoresis stains used for detection

RNA-seq analysis workflow

Sequence read genome alignments and counts reveal sites and levels of gene expression Input RNA (Total RNA, mRNA, or small RNA etc.) - fragmentation -> fragmented RNA - convert to cDNA -> DNA library -Illumina sequencing -> Mapping reads to reference genome

RNA-Seq

Sequenced-based approach Directly determine the cDNAs (RNA copies) Read count reflects abundancy of RNAs *High throughput sequencing method for transcriptome analysis*(provides global information on RNA expression patterns, relative abundance, 5' and 3' ends, splicing patterns, etc.) cDNAs generated from RNAs are sequenced using 'next generation' DNA sequencing technologies (e.g. Illumina) and reads are mapped to reference genome.

structural genomics

Sequenced-based approach to look at the structure and components of genomes and to analyze similarities between genomes

Some RNAs are translated into _________ and others are the end-products of _____ ___________

Some RNAs are translated into proteins and others are the end-products of gene expression

alternative splicing

Splicing of introns in a pre-mRNA that occurs in different ways, leading to different mRNAs that code for different proteins or protein isoforms. Increases the diversity of proteins (size of the proteome) (Selective use of multiple splice sites) ex: in different tissues

ApoB RNA editing (C to U)

Tissue-specific *deamination* of C results in two different forms of Apolipoprotein B Apolipoprotein was the first example of a mammalian RNA that undergoes editing. In apoB transcripts in the small intestine, a cytidine in the coding region is converted to a uridine generating a premature stop codon and a truncated version of the gene-encoded protein (the C to U change converts a glutamine codon (CAA) to a stop codon (UAA)).

differential RNA processing

alternative splicing or RNA editing Can result in the synthesis of more than one protein from a single gene or function as an "on-off switch" to make or not make a functional protein. *Can be regulated in a tissue-specific or developmental manner or in response to environmental stimuli.* different cell types arise and the RNA and protein content of different cell types shows considerable variation

frequency of introns in : animals, plants, and associated viruses invertebrates (e.g. Drosophila, C. elegans) lower eukaryotes (e.g. yeast) eubacteria and associated phages

animals, plants, and associated viruses - frequent invertebrates (e.g. Drosophila, C. elegans) - somewhat frequent lower eukaryotes (e.g. yeast) - rare eubacteria and associated phages - exceedingly rare

Tandem affinity purification (TAP)

double-tagging a protein and employing successive affinity purification to prepare highly purified proteins because fusing a tag to your protein may (or may not) negatively influence its behavior

uncoupled transcription and translation

eukaryotic RNAs contain introns and exons and must be edited before translation can begin in eukaryotes, while transcription occurs in the nucleus, translation occurs in the cytoplasm

RNA-mediated gene silencing pathways

eukaryotic organisms contain a system that responds to double-stranded RNA (dsRNA) to selectively trigger silencing of gene expression can act either at the post-transcriptional (PTGS) or transcriptional (TGS) levels RNAi, co-suppression, transciptional gene silencing, siRNAs, miRNAs, piRNAs

exons

expressed sequence of DNA; codes for a protein

high-throughput techniques used in functional genomics

expression profiling using DNA microarrays and RNA-seq and/or proteomics epigenomics, metabolomics, molecular interaction mapping, cellular imaging, and gene mutation

gene expression patterns are ________ and __________

gene expression patterns are *complex* and *dynamic*. *only a small % of genes in a given cell are expressed*

constitutive gene expression

gene is always expressed (mRNA transcription always occurs)

temporal/inducible gene expression

gene is expressed at specific times ex: tissue-specific/spatial, developmental, in response to environmental stimulus

developmental gene expression

genes are activated at different stages of a life cycle, ex. puberty

house-keeping genes

genes that are transcribed in all cells of the body that encode products that are required for cells maintenance and metabolism likely (constitutively) expressed in all cell types since certain proteins (and RNAs) are involved in the basic metabolic processes common to all cell types

Shotgun proteomics

identifying proteins in complex mixtures (not gel-isolated bands) using a combination of high performance liquid chromatography combined with mass spectrometry to give protein sequences

coupled transcription and translation

in prokaryotes, which lack a membrane separating the DNA from the cytoplasm, translation of mRNA molecules proceeds while they're still being transcribed; translation begins as soon as a ribosome binding site appears on the nascent mRNA; coupling of translation and transcription can give rise to polarity effects

intron

sequence of DNA that is not involved in coding for a protein is also present in the RNA copy of the gene and must be removed by a process called "RNA splicing"

Transcriptome

set of all (m)RNA molecules ("transcripts") produced from a genome Term can be applied to: complete set of transcripts for a given organism specific subset of transcripts present in a particular cell type or under specific growth conditions

proteome

set of all proteins produced under a given set of conditions Term can be applied to: complete set of proteins for a given organism specific subset of proteins present in a particular cell type or under specific growth conditions Proteome varies because it reflects genes that are actively expressed at any given time *Proteomics analyses on many samples using 2D-electrophoresis and mass spectrometry (or MS alone)* High-throughput, but less than transcriptomics

some genes are ___________ in one cell type but not another (e.g. certain __________ cells normally synthesize antibodies but _________ do not)

some genes are *expressed* in one cell type but not another (e.g. certain *immune* cells normally synthesize antibodies but *neurons* do not)

how does functional genomics relate to physiology?

studies how genomes result in cellular phenotypes and ultimately the form and function of entire organisms genome -> transcriptome -> proteome -> metabolome -> physiology

Transcriptomics

study of RNA patterns

central dogma

theory that states that, in cells, information only flows from DNA to RNA to proteins DNA -> RNA -> protein Genotype -> phenotypes

centrality of non-coding RNA

transcripts: - protein coding (2%) - non-coding (~98%) --- housekeeping --- regulatory ------ eukaryotes or prokaryotes ------ "small" (<200nt) : miRNAs, siRNAs, piRNAs, sRNAs, CRISPR, antitoxin, riboswitch ------ "long" (>200nt) : lncRNAs, XIST, H19, HOTAIR, etc

two divisions of gene control

1. Changes in Gene Content or Position 2. Changes in Gene Activity (expression patterns)

types of DNA microarray analysis

1. DNA microarray (or 'DNA chip') 2. Prepare total or (preferably) polyA+ RNA (mRNA fraction) - Grow cells under control and test condition and isolate RNA from each 3. Fluorescent cDNA (probe) production 4. Array Hybridization. 5. Detection and data analysis - laser scanning to measure the fluorescence intensity and allow simultaneous determination of relative expression levels of all genes

A to I Deamination

Adenosine Deaminase (ADAR) I is recognized as a G by translational machinery

post-transcriptional control

Determines if, how much, and when an mRNA is available for translation into a protein Many examples exist where changes in the rate of synthesis of a particular protein occur without a corresponding change in the transcription rate of the corresponding gene. Examples include where gene expression is regulated by controlling pre-mRNA processing (e.g. alternative splicing or editing), mRNA stability, and mRNA transport/subcellular localization

two approaches to transcriptome profiling

Each approach monitors the steady state levels of transcripts of each gene (a combination of both transcription (synthesis) and degradation (turnover)). 1. DNA Microarrays 2. RNA Seq

Transcriptome Profiling

Important insights into gene function can be gained by expression profiling, i.e., determining where and when particular genes are expressed. For example, some genes are switched on(induced) or switched off(repressed) by external chemical signals reaching the cell surface. *In multicellular organisms, many genes are expressed in particular cell types or at certain developmental stages.* *Furthermore, mutating one gene can alter the expression of others.* All this information helps to link genes into functional networks, and genes can be used as markers to define particular cellular states or cell types

Co-Immunoprecipitation

Macromolecular Interaction Analysis Immunopurify protein of interest (antigen) and any associated components animal cell (antigen binds the antibody) -> cell lysis -> antibody binding to protein A-agarose bead -> antigen isolation on an antibody bead -> washing and analysis (other antibodies fall off) antibody identified by mass spec

The split gene concept (mRNA splicing)

Many eukaryotic genes have their coding sequences (exons) interrupted by stretches of non-coding sequences called intervening sequences (introns)

gene silencing

mechanism of RNA interference (RNAi) : directed by ~22 RNAs whole regions of chromosomes are shut off while the same regions in other cells remain active 1. dsRNA (initiation) - dsRNA (silencing trigger) is recognized by a machinery that converts the dsRNA into 21-22 nt RNAs called siRNAs - processing -> 2. ~22nt siRNAs - siRNAs are recognized by effector complex called RISC forming an RNA/protein (RNP) complex that contains a ribonuclease activity - recognition -> 3. target mRNA - The RISC complex contains an effector nuclease SLICER responsible for destroying target RNA - degradation -> 4. genes silenced (amplification)

covalent events in RNA processing

often alters the size of RNA Modify (bases, 2'OH). Cleave (endonuclease). Trim (exonuclease, 3' to 5' or 5' to 3') Add (5' m7GpppG cap, 3' poly(A) addition, CCA,). Splice (cis vs. trans). Edit (base insertion/deletion or conversion)

piRNAs

piwi-associated RNAs (~26-33 nt in length) mainly expressed in animal *germ cells (testis/ovaries)* and protect the germline genome from active retrotransposons that can lead to genome instability processed by a largely unknown but Dicer-independent mechanism

antibodies

powerful research tools for studying proteins can make monoclonal antibodies against a protein of interest just by expressing the protein of interest, purifying it, inject into a mouse, move into tumor cells, have antibody producing hybridomas that can be used for study

mass spectrometry

proteomic analysis proteins separated by 2D electrophoresis single proteins eluted Digestion with trypsin(cuts after lysines and arginines) give fragments with unique set of sizes Sizes identified by mass spectrometry and matched to database Allows identification of unknown proteins

RT-PCR

reverse transcriptase-polymerase chain reaction A technique in which RNA is first converted to cDNA by the use of the enzyme reverse transcriptase, then the cDNA is amplified by the polymerase chain reaction.

Epigenomics

study of epigenetic component of a cell (histone modifications and DNA methylation)

metabolomics

study of metabolic expression in cells

proteomics

study of the structure and function of proteins in the human body