BMSC 320 Nucleic Acids Lecture 28 (2024) PDF

BMSC 320 Nucleic Acids Instructor: Kyle Anderson – Biochemistry, Microbiology & Immunology Lecture 28 – November 27, 2024  Review microRNAs with 7 or 8 bases of complementarity are typical – Atypical miRNAs with 6 bas...

BMSC 320 Nucleic Acids Instructor: Kyle Anderson – Biochemistry, Microbiology & Immunology Lecture 28 – November 27, 2024  Review microRNAs with 7 or 8 bases of complementarity are typical – Atypical miRNAs with 6 bases, or pair requiring some looping may still function as part of RISC, but to a lesser extent of downregulation of translation miRNAs are heavily conserved across eukaryotes – miRNAs found in worms can control similar processes in humans miRNA dysregulation can cause disease, or be a signal of disease or tissue damage Some miRNAs enhance translation (up-miRNA) Circular RNAs (circRNA) and long noncoding RNAs (lncRNA) can function as sponge of multiple miRNAs, making them unavailable to regulate their targets Eukaryotic Translation: A reminder of the basic process This module will only review the “basics” you’ve learned in previous courses so we can focus on the problems of efficiency & translating defective mRNA  Some Major Differences: Prokaryotic vs Eukaryotic Translation is coupled to transcription Translation in the cytoplasm & transcription in the nucleus 70S ribosome composed of 50S and 30S 80S ribosome composed of 60S and 40S subunits subunits Small subunit recognizes Shine-Dalgarno tRNA and small subunit scan from 5’ cap (unless sequence at 5’ end for initiation (multiple in an IRE Site exists – very rare) to a Kozak polycistronic messages) sequence containing AUG N-formyl-Methionine is the first aa Regular Methionine is the first aa mRNA is linear mRNA is held in a loop by proteins bound to cap and tail Polycistronic messages are common Polycistronic messages are extremely rare  While Ribosomes are Different, They Mostly Work the Same Prokaryote’s have ~55 ribosomal proteins while eukaryotes have ~80 Different proteins alter target sites so many antibiotics that block prokaryotic protein synthesis don’t affect eukaryotes – Compounds like cycloheximide block eukaryotic ribosomes, but not prokaryotic The human 5.8S & 28S are equivalent to the prokaryotic 23S Main ls rRNA is catalytic and main ss rRNA locates mRNA start site and checks tRNAs Export of all Nuclear RNA is Controlled Small RNAs like tRNA, miRNA associate with a few export factors snRNA’s (components of the spliceosome) are exported, associate with spliceosomal proteins & are imported again Several factors must associate with mRNAs and rRNAs to allow them to leave the nucleus  mRNA Export is Tightly Regulated Through capping the mRNA binds CBC, through tailing it binds PABP Splicing will leave Exon-Junction Complexes & SR proteins TREX = Transcription Export complex associating with RNA pol II loads onto mRNA during synthesis & processing Nuclear Export Proteins (Mex67 & Mtr2) are recruited by TREX, allow passage through NPC, are released & then return back into the nucleus All these proteins facilitate the interaction with other components  mRNA/mRNP Export is Rate-Limiting An RNA labelling assay quantifying how long an mRNA persists in the nucleus vs cytoplasm shows more time in the nucleus as export is an active and rate-limiting process – mRNA is in the nucleus ~5X longer than it persists in the cytoplasm It is theorized that the bottleneck of export may help regulate/buffer mRNA levels in the cytoplasm It also ensures only fully capped/tailed & spliced mRNAs should leave the nucleus  Translation Cycle 40S (small subunit) & initiation factors bind met-tRNA to create 43S complex 43S associates with mRNA via CBC/eIF4 – helicase activities unwind mRNA’s 5’ UTR to allow scanning for start codon Kozak sequence is used to locate the translation initiation site – Vertebrate sequence is [GCC(A/G)CCAUGG] & it varies in other eukaryotes Large subunit binds, tRNAs and elongation factors allow protein synthesis Stop codon encountered, release factors liberate protein from terminal tRNA, signal ribosome disassembly  Alternative Initiation: the IRES While 99% of human genes initiate translation through recruiting the small subunit to the 5’ end CBC via eIF4, a few hundred human genes contain Internal Ribosome Entry Sites (IRES) mRNA IRES 2o structure in the 5’ UTR binds IRES trans-acting factors (ITAF) which recruit the small subunit to a nearby AUG mRNA with IRES can be translated without a 5’ cap, or even a 5’ end in the case of some circRNAs – This strategy is more common in eukaryotic viruses than in eukaryotic genomes, but ~100 human genes utilize IRES (including about a dozen polycistronic mRNAs)  Translation & Proofreading All mRNA is eventually degraded & problematic mRNA is degraded more quickly – To help ensure undesirable mRNA’s and aberrant proteins don’t cause havoc in the cell there are methods to prevent translation of “bad” mRNA If improperly spliced or prematurely terminated mRNAs were translated, they could create truncated proteins Truncated proteins may create dominant negative proteins Enzymes which can not be turned off Proteins which permanently bind their DNA, RNA or protein partners Proteins which form non-functional dimers with good subunits Cells have 4 methods of proofreading which are utilized during translation – Nonsense mediated decay – Nonsense associated alternative splicing (less understood/less used) – Non-stop mediated decay – No-go decay