mRNA Stability Flashcards

Lecture Objectives

• Explain the causes of mRNA instability and their operation
• Explain the outcomes and relevance of mRNA instability
• Describe mRNPs and their function
• Describe the enzymes involved in prokaryotic mRNA degradation and their action
• Describe the two major pathways by which eukaryotic mRNA is degraded
• Briefly describe other specific ways mRNAs are targeted
• Describe the sequences and structures that control mRNA-specific half-lives
• Describe the quality control of mRNA from transcription to translation
• Explain the ways in which RNA is localized in the cells and the reasons for localization

20.1 Introduction

• 3′ untranslated region (UTR): The untranslated sequence downstream from the coding region of an mRNA.
• 5′ untranslated region (UTR): The untranslated sequence upstream from the coding region of an mRNA.
• stem-loop: A secondary structure that appears in RNAs consisting of a base-paired region (stem) and a terminal loop of single-stranded RNA.
• Both 3' and 5' UTRs and stem-loops are variable in size.

20.2 Messenger RNAs Are Unstable Molecules

• mRNA instability is due to the action of ribonucleases (RNAses).
• Ribonucleases differ in their substrate preference and mode of attack.
  • endoribonuclease: A ribonuclease that cleaves an RNA at an internal site(s).
  • exoribonuclease: A ribonuclease that removes terminal ribonucleotides from RNA.
• Exonucleases can be:
  • processive: An enzyme that remains associated with the substrate while catalyzing the sequential removal of nucleotides.
  • distributive: An enzyme that catalyzes the removal of only one or a few nucleotides before dissociating from the substrate.
• mRNAs exhibit a wide range of half-lives.
• mRNA decay is mRNA degradation.
• Differential mRNA stability is an important contributor to mRNA abundance, and therefore the spectrum of proteins made in a cell.
• steady state (molecular concentration): The concentration of population of molecules when the rates of synthesis and degradation are constant.

20.3 Eukaryotic mRNAs Exist in the Form of mRNPs from Their Birth to Their Death

• mRNA associates with a changing population of proteins during its nuclear maturation and cytoplasmic life.
• mRNA-protein complexes are mRNPs
• Some nuclear-acquired mRNP proteins have roles in the cytoplasm.
• A very large number of RNA-binding proteins (RBPs) exist, most of which remain uncharacterized.
• Different mRNAs are associated with distinct, but overlapping, sets of regulatory proteins, creating RNA regulons.
• The set of mRNAs that share a particular RBP is a regulon

20.4 Prokaryotic mRNA Degradation Involves Multiple Enzymes

• Occurs during the process of transcription/translation
• polyribosome (or polysome): An mRNA that is simultaneously being translated by multiple ribosomes.
• monocistronic mRNA: mRNA that codes for one polypeptide.
• Degradation of bacterial mRNAs is initiated by the removal of a pyrophosphate from the 5′ terminus.
• Monophosphorylated mRNAs are degraded during translation in a two-step cycle involving endonucleolytic cleavages, followed by 3′ to 5′ digestion of the resulting fragments.
• 3′ polyadenylation can facilitate the degradation of mRNA fragments containing secondary structure.
• 3’ stem-loop protects active mRNA from exonuclease degradation
• poly(A) polymerase (PAP): The enzyme that adds the stretch of polyadenylic acid to the 3′ end of eukaryotic mRNA.
  • It does not use a template.

20.5 Most Eukaryotic mRNA is Degraded via Two Deadenylation-Dependent Pathways

• The modifications at both ends of mRNA protect it against degradation by exonucleases (5’ cap, poly-A tail).
• Most degradation involves deadenylation of poly-A tail
• poly(A) binding protein (PABP): The protein that binds to the 3′ stretch of poly(A) on a eukaryotic mRNA protects the tail initially
• The two major mRNA decay pathways are initiated by deadenylation catalyzed by poly(A) nucleases.
• Deadenylation may be followed either by decapping and 5′ to 3′ exonuclease digestion or by 3′ to 5′ exonuclease digestion.
• The decapping enzyme competes with the translation initiation complex for 5′ cap binding.
• cytoplasmic cap-binding protein: protects the cap from decapping until translation initiation.
• Release of PABP at deadenylation also thought to destabilize cap
• The exosome, which catalyzes 3′ to 5′ mRNA digestion, is a large, evolutionarily conserved complex.
• Degradation may occur within discrete cytoplasmic particles called processing bodies (PBs).
• A variety of particles containing translationally repressed mRNAs exist in different cell types.

20.6 Other Degradation Pathways Target Specific mRNAs

• Four additional degradation pathways involve regulated degradation of specific mRNAs.
• Deadenylation-independent decapping proceeds in the presence of a long poly(A) tail.
• The degradation of the nonpolyadenylated histone mRNAs is initiated by 3′ addition of a poly(U) tail.
• Degradation of some mRNAs may be initiated by sequence- or structure-specific endonucleolytic cleavage.
• An unknown number of mRNAs are targets for degradation or translational repression by microRNAs (miRNAs).

20.7 mRNA-Specific Half-Lives Are Controlled by Sequences or Structures Within the mRNA

• Specific cis-elements in an mRNA affect its rate of degradation.
• Destabilizing elements (DEs) can accelerate mRNA decay, while stabilizing elements (SEs) can reduce it.
• AU-rich elements (AREs) are common destabilizing elements in mammals and are bound by a variety of proteins.
• Some DE-binding proteins interact with components of the decay machinery (processing bodies) and probably recruit them for degradation.
• mRNA degradation rates can be altered in response to a variety of signals.
• iron-response element (IRE): A cis sequence found in certain mRNAs whose stability or translation is regulated by cellular iron concentration.

20.8 Newly Synthesized RNAs Are Checked for Defects via a Nuclear Surveillance System

• Aberrant nuclear RNAs are identified and destroyed by an RNA surveillance system.
• The nuclear exosome functions both in the processing of normal substrate RNAs and in the destruction of aberrant RNAs.
• The yeast TRAMP complex recruits the exosome to aberrant RNAs and facilitates its 3′ to 5′ exonuclease activity.
• Substrates for TRAMP-exosome degradation include unspliced or aberrantly spliced pre-mRNAs and improperly terminated RNA Pol II transcripts lacking a poly(A) tail.
• The majority of RNA Pol II transcripts may be cryptic unstable transcripts (CUTs) that are rapidly destroyed in the nucleus.
  • Do not encode recognizable genes
  • Overlap with, and sometimes regulate, protein-coding genes
  • Sometimes arise from spurious transcription initiation

20.9 Quality Control of mRNA Translation Is Performed by Cytoplasmic Surveillance Systems

• release factor (RF): A protein required to terminate polypeptide translation to cause release of the completed polypeptide chain and the ribosome from mRNA.
• Nonsense-mediated decay (NMD) targets mRNAs with premature stop codons.
  • Can be due to nonsense mutations, splicing errors, or polymerase errors
  • Truncated proteins can interfere with other protein processes
  • Targeting of NMD-substrates requires a conserved set of UPF and SMG proteins.
• Recognition of a termination codon as premature involves unusual 3′ UTR structure or length in many organisms and the presence of downstream exon junction complexes (EJCs) in mammals.
  • In mammals, a splice junction in the 3’UTR is the signal (not usually found)
  • Yeast do not have introns, so unusually long 3’UTR is signal
• Nonstop decay (NSD) targets mRNAs lacking an in-frame termination codon and requires a conserved set of SKI proteins.
  • Prevents toxic polypeptides
  • Also releases “trapped” ribosomes
• No-go decay (NGD) targets mRNAs with stalled ribosomes in their coding regions.
  • Least understood

20.10 Translationally Silenced mRNAs Are Sequestered in a Variety of RNA Granules

• RNA granules are formed by aggregation of translationally silenced mRNA and many different proteins.
• Germ cell (maternal mRNA) granules and neuronal granules function in translational repression and transport.
  • Repressed by extensive deadenylation, activated by polyadenylation
• Processing bodies (PBs) containing mRNA decay components are present in most or all cells.
• Stress granules (SGs) accumulate in response to stress-induced inhibition of translation.

20.11 Some Eukaryotic mRNAs Are Localized to Specific Regions of a Cell

• Localization of mRNAs serves diverse functions in single cells and developing embryos.
• Three mechanisms for the localization of mRNA have been documented:
  • Uniform distribution with selective degradation
  • Free diffusion and “trapping” locally
  • Active transport of mRNA
• Localization requires cis-elements on the target mRNA and trans-factors to mediate the localization.
• zipcode (or localization signal): Any of the number of mRNA cis elements involved in directing cellular localization.
• The predominant active transport mechanism involves the directed movement of mRNPs along cytoskeletal tracks.