BMB 462 Lecture 32 Outline of Last Lecture I. Introduction to RNA processingII. 5’ cappingIII. Splicing of pre-mRNAIV. Transesterification of intronsV. Self-splicing intronsa. Group Ib. Group IIVI. Spliceosomal intronsOutline of Current Lecture I. tRNA introns – the fourth intron typeII. 3’ PolyadenylationIII. Alternative polyadenylation and splicingIV. rRNA processing in bacteriaV. rRNA processing in eukaryotesVI. snoRNPsVII. Examples of Base ModificationVIII. tRNA processingIX. Processing of small RNAsX. RNA DegradationCurrent LectureConcepts to remembers from previous courses/lectures:- Reminder from last lecture: the first 3 types of introns use transesterification reactions (Group I and Group II self-splicing introns, and Spliceosomal-associated introns)o 1 phosphodiester bond is broken and the energy is transferred to making a new phosphodiester bond (so that energy can be conserved)- Review comparison between Bacterial and eukaryotic splicing:o Only eukaryotes use snoRNPs and require export from the nucleus, because bacteria do not have a nucleus, much less a nucleolus.o Both bacteria and eukaryotes use endoRNases and exoRNases for processing, and use base modifications.These notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute.I. tRNA introns – the fourth intron typea. The fourth type of intron - tRNA introns in eukaryotes - uses a splicing mechanism that involves an endoribonuclease that cuts the RNA chain and then RNA ligation seals the exons together.i. Because the endoribonuclease and ligase activities aren't coupled, the energy from the cleavage isn't conserved and ligation requires ATP energy.II. 3’ Polyadenylationa. Note: RNA processing significantly reduces the size of the end producti. e.g. the original ovalbumin gene in the DNA is 7,700 base pairs long. Aftercapping and splicing, the mature mRNA is less than 2,000 nucleotides.b. Polyadenylation at the 3' end is analogous to the capping that occurred at the 5' end; the poly(A) tail helps protect the mRNA from degradation by exoribonucleases.c. The Poly(A) tail is made when Polymerase II comes to the end of a gene. The Poly(A) tail formation occurs at the same time as transcription termination.i. The signal in the RNA - AAUAAA - signals cleavage and polyadenylation. The sequence occurs 10-30 nucleotides before the cleavage site.ii. Once the termination signal has been read, an enzyme complex comes in and associates with CTD of the polymerase II.1. There is a GU-rich sequence found 20-40 nucleotides after the cleavage site. Here, 2 reactions from the enzyme complex occur.a. 1. An endonuclease does the actual cleavage of the RNA.b. 2. Polyadenylate polymerase (a non-templated RNA polymerase) synthesizes the run of ‘A’s on the end of the RNA in a non-templated way.iii. It has an active site that only calls for ‘A’s and is able to string them together to make a Poly(A) tail that is about 80-250 nucleotides in length for the purpose of protecting the 3' end.**No template is needed for this synthesis.iv. There are proteins that associate with the Poly(A) tail to help protect the 3' end from ribonucleases.III. Alternative polyadenylation and splicinga. The extensive processing allows an opportunity for regulation. The transcripts can be differentially processed, so that the polyadenylation and cleavage can occur at different places in different tissue types and at different times in the development of an organism. The splicing can also be different in different cell types (aka alternative splicing).i. Alternative polyadenylation and alternative splicing allow many genes to produce more than one product.1. The typical eukaryotic gene ranges from 1-5 functional products.b. Alternative polyadenylation occurs when there are 2 different potential polyadenylation sites. Depending on which one is utilized in a particular cell type,the final product can contain additional coding sequence or UTR (untranslated region).c. Calcitonin gene is a real life example of both alternative polyadenylation and alternative splicing.i. The 2 different proteins produced are calcitonin (in the 4th exon) and calcitonin-gene-related peptide (CGRP, in the 5th exon).IV. rRNA processing in bacteriaa. In bacteria, there are only 3 rRNAs, but there are 7 rRNA operons with a variable number of tRNAs interspersed.i. Pictured is a single pre-rRNA transcript with all 3 rRNAs and a tRNA.b. First, bases in the rRNA and tRNA are modified (i.e. by methylation, often of the 2' OH, or conversion to pseudouridine/dihydrouridine)c. Then cleavage by endoribonucleases occurs.i. EndoRNase II cleaves and makes the 5' end of tRNA. It is also called RNaseP.d. Further nucleases, exoribonucleases, then chew the ends of the fragments to cleave off remaining introns to form the completed mature RNAs.e. There are ribosomal proteins associated with the rRNA throughout this process.V. rRNA processing in eukaryotesa. In eukaryotes, 3 rRNAs are contained within one transcript and the fourth is on another.b. The first three rRNAs are transcribed by polymerase I. The primary transcript is associated with some rRNAs that assemble with it. These make up the preribosome.i. This assembly occurs in the nucleolus.c. Then snoRNPs (small nucleolar RNPs) help with the processing of the rRNA. These are analogous to the snRNPs.i. Much like in bacteria, there are modifications of the bases, initial cleavages of RNA by endoRNases to break apart the different rRNAs.ii. Then a 5S rRNA (a polymerase III product) comes in and begins to make the pre-40S and 60S ribosomal parts.1. You start with the whole 18S rRNA plus a bunch of ribosomal proteins; that will be the 40S (the small subunit) of the eukaryotic ribosome.2. The other 3 rRNAs along with ribosomal proteins make the 60S (larger) ribosomal subunit.iii. Additional cleavages are performed by exoribonucleasesd. The cell now has 2 fairly large ribosomal complexes, the pre-small and pre-large subunits, which it has to remove from the nucleus and get out in the cell so that the subunits could finally come together and function.i. They must remain nonfunctional in the nucleus so that they don't start translating unspliced messages or other things found in the nucleus; they should only be functional in the cytoplasm.ii. Thus there is additional processing in the cytosol that must occur to makethe ribosomes fully functional.VI.
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