Post-transcriptional regulation of satellite cell quiescence by TTP-mediated mRNA decay.

Skeletal muscle satellite cells in their niche are quiescent and upon muscle injury, exit quiescence, proliferate to repair muscle tissue, and self-renew to replenish the satellite cell population. To understand the mechanisms involved in maintaining satellite cell quiescence, we identified gene transcripts that were differentially expressed during satellite cell activation following muscle injury. Transcripts encoding RNA binding proteins were among the most significantly changed and included the mRNA decay factor Tristetraprolin. Tristetraprolin promotes the decay of MyoD mRNA, which encodes a transcriptional regulator of myogenic commitment, via binding to the MyoD mRNA 3' untranslated region. Upon satellite cell activation, p38α/ß MAPK phosphorylates MAPKAP2 and inactivates Tristetraprolin, stabilizing MyoD mRNA. Satellite cell specific knockdown of Tristetraprolin precociously activates satellite cells in vivo, enabling MyoD accumulation, differentiation and cell fusion into myofibers. Regulation of mRNAs by Tristetraprolin appears to function as one of several critical post-transcriptional regulatory mechanisms controlling satellite cell homeostasis.

hnRNP F complexes with tristetraprolin and stimulates ARE-mRNA decay.

The tristetraprolin (TTP) family of zinc-finger proteins, TTP, BRF1 and BRF2, regulate the stability of a subset of mRNAs containing 3'UTR AU-rich elements (AREs), including mRNAs coding for cytokines, transcription factors, and proto-oncogenes. To better understand the mechanism by which TTP-family proteins control mRNA stability in mammalian cells, we aimed to identify TTP- and BRF1-interacting proteins as potential TTP-family co-factors. This revealed hnRNP F as a prominent interactor of TTP and BRF1. While TTP, BRF1 and hnRNP F are all RNA binding proteins (RBPs), the interaction of hnRNP F with TTP and BRF1 is independent of RNA. Depletion of hnRNP F impairs the decay of a subset of TTP-substrate ARE-mRNAs by a mechanism independent of the extent of hnRNP F binding to the mRNA. Taken together, these findings implicate hnRNP F as a co-factor in a subset of TTP/BRF-mediated mRNA decay and highlight the importance of RBP cooperativity in mRNA regulation.

Phosphorylation of tristetraprolin by MK2 impairs AU-rich element mRNA decay by preventing deadenylase recruitment.

mRNA turnover is a critical step in the control of gene expression. In mammalian cells, a subset of mRNAs regulated at the level of mRNA turnover contain destabilizing AU-rich elements (AREs) in their 3' untranslated regions. These transcripts are bound by a suite of ARE-binding proteins (AUBPs) that receive information from cell signaling events to modulate rates of ARE mRNA decay. Here we show that a key destabilizing AUBP, tristetraprolin (TTP), is repressed by the p38 mitogen-activated protein kinase (MAPK)-activated kinase MK2 due to the inability of phospho-TTP to recruit deadenylases to target mRNAs. TTP is tightly associated with cytoplasmic deadenylases and promotes rapid deadenylation of target mRNAs both in vitro and in cells. TTP can direct the deadenylation of substrate mRNAs when tethered to a heterologous mRNA, yet its ability to do so is inhibited upon phosphorylation by MK2. Phospho-TTP is not impaired in mRNA binding but does fail to recruit the major cytoplasmic deadenylases. These observations suggest that phosphorylation of TTP by MK2 primarily affects mRNA decay downstream of RNA binding by preventing recruitment of the deadenylation machinery. Thus, TTP may remain poised to rapidly reactivate deadenylation of bound transcripts to downregulate gene expression once the p38 MAPK pathway is deactivated.

A tethering approach to study proteins that activate mRNA turnover in human cells.

The regulation of mRNA turnover occurs in part through the action of mRNA-binding proteins that recognize specific nucleotide sequences and either activate or inhibit the decay of transcripts to which they are bound. In many cases, multiple mRNA-binding proteins, including those with opposing functions, bind to the same RNA sequence. This can make the study of the function of any one of these proteins difficult. Furthermore, monitoring endogenous mRNA decay rates using drugs that inhibit transcription (e.g., actinomycin D) can introduce pleiotropic effects. One way to circumvent these problems is to tether the protein of interest (POI) through a heterologous RNA-binding domain to an inducible reporter mRNA and measure the effect of the bound protein on mRNA decay. In this chapter, we illustrate the use of the tethering technique to study the role of a particular mRNA-binding protein, TTP, on the decay of an otherwise stable mRNA to which it is tethered through a fusion to the bacteriophage MS2 coat protein.

An unconventional human Ccr4-Caf1 deadenylase complex in nuclear cajal bodies.

mRNA deadenylation is a key process in the regulation of translation and mRNA turnover. In Saccharomyces cerevisiae, deadenylation is primarily carried out by the Ccr4p and Caf1p/Pop2p subunits of the Ccr4-Not complex, which is conserved in eukaryotes including humans. Here we have identified an unconventional human Ccr4-Caf1 complex containing hCcr4d and hCaf1z, distant human homologs of yeast Ccr4p and Caf1p/Pop2p, respectively. The hCcr4d-hCaf1z complex differs from conventional Ccr4-Not deadenylase complexes, because (i) hCaf1z and hCcr4d concentrate in nuclear Cajal bodies and shuttle between the nucleus and cytoplasm and (ii) the hCaf1z subunit, in addition to rapid deadenylation, subjects substrate RNAs to slow exonucleolytic degradation from the 3' end in vitro. Exogenously expressed hCaf1z shows both of those activities on reporter mRNAs in human HeLa cells and stimulates general mRNA decay when restricted to the cytoplasm by deletion of its nuclear localization signal. These observations suggest that the hCcr4d-hCaf1z complex may function either in the nucleus or in the cytoplasm after its nuclear export, to degrade polyadenylated RNAs, such as mRNAs, pre-mRNAs, or those RNAs that are polyadenylated prior to their degradation in the nucleus.

Identification of pentatricopeptide repeat proteins in Trypanosoma brucei.

A new class of organellar proteins, characterized by pentatricopeptide repeat (PPR) motifs, has been identified in plants. These proteins contain multiple 35-amino acid repeats that are proposed to form a super helix capable of binding a strand of RNA. All PPR proteins characterized to date appear to be involved in RNA processing pathways in organelles. Twenty-three PPR proteins have been identified in Trypanosoma brucei and database research indicates that most of these proteins are predicted to contain the traditional mitochondrial target sequence. Orthologues of each of the 23 proteins have also been identified in Leishmania major and Trypanosoma cruzi, indicating that these proteins represent a highly conserved class of proteins within the kinetoplastid family. Preliminary experiments using RNAi to specifically silence one identified PPR gene (TbPPRl- Tb927.2.3180), indicate that cells depleted of TbPPRl transcripts show a slow growth phenotype and altered mitochondrial maxicircle RNA profiles. This initial characterization suggests that PPR proteins will play important roles in the complex RNA processing required for mitochondrial gene expression in trypanosomes.

An intragenic guide RNA location suggests a complex mechanism for mitochondrial gene expression in Trypanosoma brucei.

In Trypanosoma brucei, two classes of transcripts are produced from two distinct mitochondrial genome components. Guide RNAs (gRNAs) are usually minicircle encoded and exist as primary transcripts, while the maxicircle-encoded rRNAs and mRNAs are processed from a polycistronic precursor. The genes for the gRNAs gMURF2-II and gCYb(560) each have uncommon kinetoplast DNA (kDNA) locations that are not typically associated with transcription initiation events. We demonstrate that the conserved maxicircle gRNA gMURF2-II has an unusual location within the ND4 gene. This is the first report of a completely intragenic gene in kDNA. In addition, the gMURF2-II and ND4 transcripts are generated by distinctly different events; the ND4 mRNA is processed from a polycistronic precursor, while transcription of the gRNA initiates downstream of the 5' end of the ND4 gene. The gCYb(560) gene has an atypical minicircle location in that it is not flanked by the inverted repeat sequences that surround the majority of minicircle gRNA genes. Our data indicate that the mature gCYb(560) gRNA is also a primary transcript and that the 5'-end heterogeneity previously observed for this gRNA is a result of multiple transcription initiation sites and not of imprecise 5'-end processing. Together, these data indicate that gRNA genes represent individual transcription units, regardless of their genomic context, and suggest a complex mechanism for mitochondrial gene expression in T. brucei.