MBIO 4700 - Topic 3

RNA 

The work hourse of nucleic acids (since DNA only stores info, RNA has to do everything else)

RNA does not just float around, it ____

Folds 

Different RNA bonds

It has an extra H-bond between GU, and then the regular AU and GC. 

RNA can form…

Secondary and tertiary structures

RNA can fold into _____ ways

Different 

Why should we care about the folding of RNA?

They cannot function properly unless they fold properly

Different ways RNA folding is involved in gene regulation

1.) Transcriptional and translational stop
2.) Speed of translation
3.) Riboswitches
4.) Ribozymes 

Transcriptional and translational stop in RNA 

It forms a loop, which acts as a stop signal to stop transcription or translation

Speed of translation (complex folds)

mRNA have complex folding patterns, which regulates how fast the mRNA is translated and how fast the peptides fold

Riboswitches

Involves metabolites, as well as small RNAs like miRNAs and siRNAs, which can inhibit translation from occuring

Ribozymes

They are not built into the sequence for regulation, but instead for catalytic activity 

RNA world 

Proposes that RNA came first and DNA came later

Rfam database 

It curates different types of RNA molecules and allows us to see which RNA family they belong to, as well as what function they may have

Small noncoding RNAs (examples)

1.) siRNA 
2.) miRNA
3.) PIWI piRNA 

siRNAs

Small interferring RNAs derived from double stranded RNA (transcribed from a target gene) and is a perfect match to the mRNA or target RNA 

miRNAs

1.) Encoded by miRNA genes 
2.) They are invovled in translation suppression and mRNA decay

miRNA matching mRNAs

They do not need to be a perfect match, therefore one miRNA can target several mRNA

PIWI piRNA 

A type of rasiRNA (repeat associated small interfering RNAs), which is RNA derived from repeat rich or transposon rich regions of the genome

PIWI piRNA function

Supresses gene expression in germ line tissue

Function of siRNA and miRNAs

1.) Protects you from viruses
2.) Knock down genes (inactivate)

In order to identify ribozymes from linear sequences, you have to…

Fold it 

Vienna notation

A way of annotating RNA sequences, to show how they fold 

Vienna notation components

() = base pairing 
….. = loops
---- = insertions/delection (or missing data) 

RNA is assigned to RNA families based on…

Folding patterns 

Predicting RNA folding patterns process

Find base pairs along the string that are correct or optimal, by maximizing hydrogen bonding between base pairs and minimizing free energy (based on best overall free energy state)

What's the problem with the strategy of maximizing H-bodning and minimizing free energy?

You get multiple possible structures, therefore you may need to do some manual adjustments 

New generation of programs that looks at RNA folding

In addition to maximizing H-bonding and minimizing free energy, they are using covariance analysis, which uses models to determine what RNA molecules may look like

What is GALAXY?

It does things that are related to 'omics

GALAXY sites

It has several sites across the world, the best being the European and French sites, and the worst being the main American site

RNA central 

Still in its early stages, but it has various tools that allows you to explore RNA molecules 

R2DT

It takes a sequence and folds it based on a template you give it 

Comparative RNA website (CRW)

1.) Very old
2.) Does not have tools embedded in it
3.) It allows you to download things onto the database, but it can't do anything with it since it has no tools

CRW vs. RNA central 

1.) RNA central has tools embedded in it, as well as databases you can upload your information into
2.) CRW only allows you to upload you data 

Potential problem with R2DT

Still have to manually check what you get back because it will have certain biases, such that it may miss some unique structures that are less common or that are not present in the template 

Rfam also has access to…

R2DT (because it is embedded within RNA central, despite being an independant website)

Rfam also has something more advanced…

Infernal, which looks at folding possibilities using a probability criteria, based on covariance models 

Mfold is now known as 

UNAfold 

UNAfold/Mfold

The grand program that started RNA folding, as it predicts RNA and DNA secondary structures using nearest neighbour thermodynamic rules 

What UNAfold take into account when making predictions 

1.) H-bonding
2.) Van der waals interactions 
3.) Hydrophobic regions 

UNAfold assumption about predictions

1.) Nucleic acids form a helical configuration and like to be double stranded
2.) Base pairs like to stack on top of each other 

Creator of Mfold 

Michael Zuker

Warning when using UNAfolder 

1.) The predicted structure may not be the actual biological structure
2.) May get back many structures that are thermodynamically possible 

Conditions that can be modified in UNAfold

1.) Temp
2.) pH
3.) Salt concentrations

Pseudoknots

It's when a region of the RNA molecule interacts with another region further down on the same molecule

UNAfold can't predict…

Pseudoknots 

Why do we care about pseudoknots

1.) Sometimes embedded in transcripts, therefore it can play a part in gene regulation
2.) It can form the catalytic sites of ribozymes
3.) It can promote frame-shifting, such that it causes ribosomes to skip a nucleotide

""

One arc of interactions overlap with another arc of interactions, which is what RNA folding tools struggle with

What strategy is usually used when using UNAfold 

1.) Analyze the sequence in small segments and check literature for known structures
2.) Don't be afraid to fold by hand
3.) Note that some long-range interactions may be missed by the program (unless you set it as a “constraint”)

Constraint

It's when you tell the UNAfold program what you already know about the molecule and its structure, which allows you to narrow down the amount of output it generates

Sequence format used in UNAfold 

You can use FASTA format with RNA and even DNA, as it will autimatically convert it into an RNA sequence

ITS sequences

Internal transcribed spacer sequences, which separate the 5.8S rRNA gene from the rRNA genes of SSU and LSU

Are spacers introns?

Even though they are removed during processing, they are not considered introns since they do not get spliced out

How are ITS sequences removed

They are removed by specific enzymes 

ITS folded structure

It forms a hairpin

Why are ITS regions important

It brings the rRNA genes together, allowing them to be readily removed 

Conservation of ITS sequences

Their sequences are highly variable across species (i.e. not conserved), buts hairpin structure is highly conserved

The constraints you give UNAfold are based on…

Based on comparative analyses on other structures

Circular structure plot

Just another way of visualizing your RNA structure

RNA structure logo (requirements)

1.) Need an alignment of multiple sequences
2.) Have to know a little about the molecule you're working on
3.) The alignment must be coded in the Vienna notation

RNA structure logos show a lot of…

It shows a lot of variability, usually due to compensatory mutations 

Compensatory mutations representation in RNA structure logos 

"It shows an M "

Compensatory mutations in RNA

A change in one position causes a change in another position, in order to maintain an interaction between the two positions

"Upside down letters"

It means they appear less frequently than expected in the alignment 

Mutual information theory

Looks at mutual dependence between variables (can be linear or non-linear dependence) and tries to quantify the amount of information you can conclude from one variable by looking at the other variable 

Mutual information theory in simple terms

What can you conclude about Y, based on what happened to X?

What was observed in the structure logo of the rDNA ITS sequences

1.) They always form a hairpin
2.) There are quite a few parts that are highly conserved

Compensatory substitutions in RNA occur due to..

Functional/structural constraints

Observation in the rDNA ITS phylogenetic tree

It was observed that the sizes of ITS1 evolved along with ITS2, such that when a change in the size of ITS1 was observed, a change also occured in the size of ITS2 

Overall observations when looking at ITS sequences

1.) IST1 and 2 coevolve in regards to size
2.) Conservation of the structure is supported by compensatory substitutions

Application of studying ITS sequences

It helps understand the function of ITS, allowing us to assess the utlity of ITS regions for molecular taxonomy 

Stockholm format 

1.) Format being used for RNA alignments in newer tools, including Rfam
2.) It shows annotation features along the alignment

Features have to be added in the stockholm format _______

Manually 

Infernal 

A program that uses structural annotated RNA alignments to develop covariance models, which can then be used to search genomes for RNA elements, based on recognizing RNA folds 

Infernal when looking at non-coding RNAs

The primary sequence conservation is low, but there is conservation at the structural level

Covariance is a measure of _______ ___________ between two random variables

Linear dependence 

How does infernal work

1.) Align sequences and find potential H-bonding interactions 
2.) Look for covariation

What is covariation?

It's similar to compensatory substitutions, such that covariance analysis is optimized for looking at compensatory events 

Compensatory substitutions/covariation is what allows RNA families to be highly _ in sequences

Diverse 

Infernal/covariation analysis basically assesses your alignment for…

Compensatory changes

Compensatory subsitutions are informative with regards to _

Structures 

RNA folding programs (other than UNAworld)

RNA structure 

RNA structure 

1.) User friendly
2.) Claims to do pseudoknots
3.) Uses alignment to generate structures

RNAstrcuture is linked to…

Matthews lab, which has all kinds of RNA web servers that it is connected to

Problem with RNAstructure

1.) Formatting is an issue
2.) It can't have any non-nucleotide characters, such as numbers
3.) The upper and lower case letters have meaning 

Upper and lowercase letter meanings in RNAstructure

Upper = Not folded/single stranded
Lower = Folded

UNAfold vs. RNAstructure

Format it not a problem in UNAfold, but it is in RNAstructure

RNAstructure works better if…

If you already have an alignment available for the gene of interest, as the program can use that info to asssist in folding

ViennaRNA web services

1.) Predominant in Europe
2.) Provides a list of tools
3.) Best place to start if you want to fold RNA molecules

ViennaRNA vs. RNAstructure

ViennaRNA is better

ViennaRNA and UNAfold

They are similar, in that it folds RNA molecules, but allows you to add in constraints

DrFORNA

A good visualization tool for the folding of an RNA molecule, embedded within ViennaRNA

DrFORNA is better when..

1.) When you have a smaller molecule
2.) Unless, you have already have a defined folding pattern that was done in another program, and you want to use this program to get better visualization

Jalview

1.) Free
2.) Updated and curated
3.) Has lots of fundamental tools to deal with DNA, RNA, and proteins
4.) Is linked to other sites
5.) User-friendly