Functional Diversity of RNA molecules

• Central dogma of biology → DNA makes RNA which makes proteins
• RNA plays more than messenger role, important for agent that translate mRNA into proteins + transfer RNA which adapts DNA-RNA base-pairs for proteins
• RNA transmits information and carries out all cellular functions → RNA can carry out enzymatic reactions and can translate RNA into protein
• RNA can also store information → viruses transmit genome using RNA

• in DNA reduplication, ribosome is RNA itself, also additional functions of rna

• primer rna and telomerase RNA used in reduplication to start copying more DNA after you reach end of chromosome

• in transcription, splicing is done with regulation and transcription processes including SlowRNA, XisRNA, large non-coding RNAs that coat X-chromosome to repress it

• microRNA and small-interferingRNA which are used for degredation

  • gene regulation and expression is determined by these rnas post-transcriptionally (done in cytoplasm once mRNA has been translocated outside of nucleus so that it doesn't get translated into protein)
  • RNAi pathway → mRNA has already been synthesized, but we want to inhibit expression
    • in DNA, there is gene that encodes miRNA, RNA polymerase binds to DNA and then you get expression of gene, then you get RNA transcript (miRNA is non-coding)
    • in certain regions, the RNA in miRNA has complementary bindings and forms hair-pins → the final structure is 1 hairpin with a poly-A region and then starts with a CAP
      • this is known as primary miRNA or pri-miRNA → going to be trimmed with DGCR8 and Drosha → enzyme complex trims brances off of hairpin with poly-A region and the CAP
      • then you get, pre-miRNA which is just the hairpin
    • after, the pre-miRNA is exported out of the nucleus through EXP5 into cytoplasm
    • then in cytoplasm, RISC-loading complex (has multiple proteins including DICER and TRBP) is going to be bound to pre-miRNA by these two proteins
    • the loop at the end of the hairpin is going to be gone, then you get a double-stranded mi-RNA which is your duplex
    • then the argonaute protein, which is a helicase, unzips the two strands apart in the duplex (no double-stranded RNA can bind to mRNA) so it can bind to mRNA
      • one of those strands gets loaded onto RISC protein with single-strand miRNA
      • the RISC complex has siRNA, then finds mRNA that has complementary to siRNA
      • if the complex finds the mRNA with perfect complement to siRNA, RISC complex will destroy mRNA so it doesn't get expressed by ribosome (helps repress expression of gene at post-transcriptional level)
      • else, if the mRNA is not complementary to the siRNA but has high enough degree of complemetary, the RISC complex sticks to mRNA and prevents ribosome from binding
  • ribosomal RNA is what the Ribosome catalytic core is made up of → in evolution, rRNA was passed on and all living organisms taht had it would have larger diversity of functions and all other organisms died
  • all species have ribosome that is similar to every other, structure shows that the peptide bond formation catalyzed by an anderine in rRNA + protein components that are not RNA only play structural or auxillary role
  • the reason RNA can carry out these functions is because it can be double stranded and fold + base pair with itself
  • splicing RNA is at core of DNA to protein path → have the ability for mRNA to excise away introns and maintain exons to create new proteins
    • spliceosome core is made of snRNAs
    • RNA cna replicate itself → for some ribozymes
    • RNA has a U shape which allows it to fold more easily with at least one turn of an RNA helix
  • RNA can mediate enzymatic activities which means it can catalyze reactions to itself and on other RNAs
    • self-cleaving ribozymes
    • pre-tRNA cleavage by RNaseP
    • Self-splicing by group I introns
    • Capping reaction similar to branching reactino of group II introns
    • self-splicing of group II introns by branching reaction
    • Hydrolytic self-splicing of group II introns

  

  • RNA also has something called Riboswitches where RNA structures can undergo self-regulatory conformational change by sensing and resopnding directly to the environment

  • examplse are sensing environmental stimuli directly without proteins + RNA conformational changes effect gene activity

    

  • ribosome catalytic core made netirely of RNA: ribozymes → rRNA is at the core of DNA-to-protein path as a part of translation + ribosome acted as an evolutionary stressor → even peptide bond formation is catalyzed by an adenine in rRNA during translation → proteins in the ribosome play mostly auxiliary roles

  • folding allows for double-stranded RNA, and can even base pair with itself → at 5' end, certain regions that are complementary form partial double helices and continues to do so → different energy states and base pairing creates a lot of structural diversity in the folding for structure (at 2d and 3d level)

    • another benefit of RNA is the Uracil nucleotide which can bind to both A and G
    • RNA can also mediate enzymatic activites without using up itself → this involves things like self-cleaving ribozymes, pre-tRNA cleavage by RNaseP, self-splicing by group I introns, capping reaction similar to branching reacitno of group II introns, self splicing of group II introns by a branching reaction, and hydrolytic self-splicing of group II introns
    • as an engineer, you can design folds, synthesize RNA moleucles, and then apply that to a tertiary fold and thus catalyze reactions by effectively designing the strucutre based on the fold
    • a design element for complementary base pairs is that they can change under certain conditions, makes RNA versatile but also predictable and adds much more diversity → this is how riboswitches work fundamentally
      • in riboswitches, when a molecule is sensed, it can change a riboswtich and expose translationational start and launch a process without any factors intervening

    

  • snRNA at core of splicing → the small rnas used for catalysis of splicing and recognize splice sites + interact with other proteins

  • another benefit of RNA is that it can replicate by itself → ribozymes for example act as RNA dependent RNA polymerase

  • why is this? RNA preceeds DNA and protein machinery, in fact it was RNA makes RNA which makes RNA (self-modification, folding, and final replication) → selected template binding domains can even replicate the helixes and folds of RNA beyond just the independent sequences

  • RNA carried information, it also acted as transfer molecule, and also carried out reactoins and folding (could do multiple processes at the same time) → led to specialization later on with DNA to RNA to proteins

  • How did we get there? pre-life had clay minerals that guided synthesis of single RNA strands → used to be spontaneously created, then detached, folded, and catalyzed

    • then self/cross replication occured with different favourings/selection → that allowed for modifications in how bonding worked and self-replication → led to sensing, catalysis, and other complex functions under the pre-defined structure of RNA and its interaction with its surroundings

  • viruses and evolution probably played a major role in how RNA specialized to DNA and proteins for information storage and whatnot

    • evolution continues to this day → young noncoding RNAs also have additional regulatory layers involved in complex organisms (an example of this is microRNA which is very very recent) and is a layer of life that evolution will explore

  ---

  RNA Transcript Reconstruction:

  • Gene expression analysis is conducted by two primary technologies (Microarray tech & RNA-seq tech):
    1. DNA Microarray assay tech → captures expression of genes in ENTIRE genome + what proteins are produced based on gene expression + what is happening in particular cell and does this by identifying mRNAs made by cell during transcription
       1. isolate mRNAs from a cell, then perform reverse transcriptase polymerase chain reaction → turns mRNA into complementary DNA (cDNA) so now mRNA becomes template
       2. mRNAs have poly-A tail, so primer of poly-T is used to get reverese transcriptase going, then another enzyme is used for RNA degredation
       3. DNA polymerase then begins on cDNA as it acts as the template, thus synthesizing the complement to the cDNA strand
          1. the double-stranded DNA itself is called cDNA, and can be used for tools like Gel Electropheresis
       4. the microarray assay has several wells that each have a single-stranded DNA probe which represents a particular gene
          1. the grid can be thought of as many genes in an organism
       5. the cDNA are labelled with flourescent tags and introduced to array, and then put in the wells → you can add multiple different types of probes to the same well to compare different samples or if you're looking for several different genes
       6. the cDNA can hybridize with only complementary pairs, thus the DNA probe must be complementary to the cDNA in order for the process to occur
       7. at the end, the total number of colored cells indicates the expression of the target gene, and different flourescene levels indicate activity across the entire assay (cDNA with little to no binding is washed away)
       • advantage of technique is that is can focus on small regions, even if there are a few molecules or cell samples
    2. RNA-seq technology: short reads of mRNA are sequenced and mapped to the genome → this is done by extracting RNA and then barcoding the samples, and then reading the barcodes provides expression levels

       1. this is done at the cellular level to compare gene expression levels amongst different cells/tissue samples → you can count the barcodes relative to the number of cells and the number of genes (CELL0LEVEL COVARIATES and GENE-LEVEL COVARIATES)

       2. cells from suspension get paired with beads, the oil coats the beads and cells togehter and forms an emulsion

       3. each bead has barcoded dt-oligocodes and captures mRNA through RNA hybridization

       4. you then pull out the beads and conduct reverse transcription process with template switching to create cDNAs associated with the same barcode from before

       5. the cDNA is then amplified with PCR and is then ready for sequence analysis, after which each mRNA is mapped to its cell of origin and gene of origin based on the barcode

       6. once mRNA is collected, contaimnant DNA and rRNA is removed, and after the purification, you can reverse-transcribe cDNA and then add ligate sequence adaptors for PCR amplification

       7. the sequencing machine only sequences the cDNA ends becasue sequencing long RNA or DNA is difficult (this is because the sequencing reaction was based on ability to distinguish between transcripts of different lengths)

          

          

          ---

          • you can predict the next nucleotide because of the length of the transcript + test this with specific reactions to each nucleotide
          • the lengths helped you identify when to start and stop the reaction + evaluate the final output with a 900-1000 base pair length
          1. NGS allowed for having reactions across a 2-D plane, and then imaging allows you to look at each individual nucleotide without any real organization → different cycles of reactions allow for higher resolution and much more efficient output (final step of sc-RNAseq)
    • the computational challenge of understanding the RNA/transcriptome is to use these small sequenced cDNA tails to reconstruct the transcriptome → new systems are being made with long RNA sequence reactions such as nanopore
    • with a set of reads, you can align the reads to the genome, and then assemble the transcripts from the spliced alignments OR assemble transcripts directly through a different method apart from sequence alignment (essentially more direct methods) without aligning reads to a and then align transcripts to genome database/library

RNA Folding

• RNA structure has 3 layers → primary, secondary, tertiary → complementary base pairs leads to bonds, opposing base pairs create loops in secondary sequence → tertiary fold based on biophysical constraints (tertiary is hard and requires simulation)
• RNA secondary structure: