Joel G. Belasco

Let Me Count the Ways: Mechanisms of Gene Regulation by miRNAs and siRNAs

The downregulation of gene expression by miRNAs and siRNAs is a complex process involving both translational repression and accelerated mRNA turnover, each of which appears to occur by multiple mechanisms. Moreover, under certain conditions, miRNAs are also capable of activating translation. A variety of cellular proteins have been implicated in these regulatory mechanisms, yet their exact roles remain largely unresolved.

The bacterial enzyme RppH triggers messenger RNA degradation by 5′ pyrophosphate removal

The long-standing assumption that messenger RNA (mRNA) degradation in Escherichia coli begins with endonucleolytic cleavage has been challenged by the recent discovery that RNA decay can be triggered by a prior non-nucleolytic event that marks transcripts for rapid turnover: the rate-determining conversion of the 5′ terminus from a triphosphate to a monophosphate. This modification creates better substrates for the endonuclease RNase E, whose cleavage activity at internal sites is greatly enhanced when the RNA 5′ end is monophosphorylated. Moreover, it suggests an explanation for the influence of 5′ termini on the endonucleolytic cleavage of primary transcripts, which are triphosphorylated. However, no enzyme capable of removing pyrophosphate from RNA 5′ ends has been identified in any bacterial species. Here we show that the E. coli protein RppH (formerly NudH/YgdP) is the RNA pyrophosphohydrolase that initiates mRNA decay by this 5′-end-dependent pathway. In vitro, RppH efficiently removes pyrophosphate from the 5′ end of triphosphorylated RNA, irrespective of the identity of the 5′-terminal nucleotide. In vivo, it accelerates the degradation of hundreds of E. coli transcripts by converting their triphosphorylated 5′ ends to a more labile monophosphorylated state that can stimulate subsequent ribonuclease cleavage. That the action of the pyrophosphohydrolase is impeded when the 5′ end is structurally sequestered by a stem-loop helps to explain the stabilizing influence of 5′-terminal base pairing on mRNA lifetimes. Together, these findings suggest a possible basis for the effect of RppH and its orthologues on the invasiveness of bacterial pathogens. Interestingly, this master regulator of 5′-end-dependent mRNA degradation in E. coli not only catalyses a process functionally reminiscent of eukaryotic mRNA decapping but also bears an evolutionary relationship to the eukaryotic decapping enzyme Dcp2.

All Things Must Pass: Contrasts and Commonalities in Eukaryotic and Bacterial mRNA Decay

Despite its universal importance for controlling gene expression, mRNA degradation was initially thought to occur by disparate mechanisms in eukaryotes and bacteria. This conclusion was based on differences in the structures used by these organisms to protect mRNA termini and in the RNases and modifying enzymes originally implicated in mRNA decay. Subsequent discoveries have identified several striking parallels between the cellular factors and molecular events that govern mRNA degradation in these two kingdoms of life. Nevertheless, some key distinctions remain, the most fundamental of which may be related to the different mechanisms by which eukaryotes and bacteria control translation initiation.

Regulation of proto-oncogene mRNA stability

IV. Mechanisms of degradation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Deadenylation precedes decay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Translational coupling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. A U r i c h m R N A binding proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

CCR4-NOT Deadenylates mRNA Associated with RNA-Induced Silencing Complexes in Human Cells

ABSTRACT MicroRNAs (miRNAs) repress gene expression posttranscriptionally by inhibiting translation and by expediting deadenylation so as to trigger rapid mRNA decay. Their regulatory influence is mediated by the protein components of the RNA-induced silencing complex (RISC), which deliver miRNAs and siRNAs to their mRNA targets. Here, we present evidence that CCR4-NOT is the deadenylase that removes poly(A) from messages destabilized by miRNAs in human cells. Overproducing a mutationally inactivated form of either of the catalytic subunits of this deadenylase (CCR4 or CAF1/POP2) significantly impedes the deadenylation and decay of mRNA targeted by a partially complementary miRNA. The same deadenylase initiates the degradation of “off-target” mRNAs that are bound by an imperfectly complementary siRNA introduced by transfection. The greater inhibitory effect of inactive CAF1 or POP2 (versus inactive CCR4) suggests a predominant role for this catalytic subunit of CCR4-NOT in miRNA- or small interfering RNA (siRNA)-mediated deadenylation. These effects of mi/siRNAs and CCR4-NOT can be fully reproduced by directly tethering RISC to mRNA without the guidance of a small RNA, indicating that the ability of RISC to accelerate deadenylation is independent of RNA base pairing. Despite its importance for mi/siRNA-mediated deadenylation, CCR4-NOT appears not to associate significantly with RISC, as judged by the failure of CAF1 and POP2 to coimmunoprecipitate detectably with either the Ago or TNRC6 subunit of RISC, a finding at odds with deadenylase recruitment as the mechanism by which RISC accelerates poly(A) removal.

A structural model for the HIV-1 Rev-RRE complex deduced from altered-specificity rev variants isolated by a rapid genetic strategy.

A broadly applicable genetic strategy was developed for investigating RNA-protein interactions and applied to the HIV-1 Rev protein. By rapidly screening thousands of Rev-RNA interactions in Escherichia coli, we isolated Rev suppressor mutations that alleviated the deleterious effect of mutations in RRE stem-loop IIB, the high affinity RNA-binding site for Rev. All of these suppressor mutations map to a single arginine-deficient face of a Rev alpha-helix, and some alter the binding specificity of the protein, providing genetic evidence for direct contacts between specific Rev amino acids and RNA nucleotides in the RNA complex of Rev. The spatial constraints suggested by these data have enabled us to model the structure of this complex.

Structural model for the cooperative assembly of HIV-1 Rev multimers on the RRE as deduced from analysis of assembly-defective mutants

The functional efficacy of the HIV-1 Rev protein is highly dependent on its ability to assemble onto its HIV-1 RNA target (the RRE) as a multimeric complex. To elucidate the mechanism of multimeric assembly, we have devised two rapid and broadly applicable strategies for examining cooperative interactions between proteins bound to RNA, one based on cooperative translational repression of a two-site reporter and the other on gel shift analysis with crude E. coli extracts. Using these strategies, we have identified two distinct surfaces of Rev (head and tail) that are critical for different steps in multimeric assembly. Our data indicate that Rev assembles cooperatively on the RRE via a series of symmetrical tail-to-tail and head-to-head protein-protein interactions. The insights into molecular architecture suggested by these findings have enabled us to derive a structural model for Rev and its multimerization on the RRE.

Biological consequences of segmental alterations in mRNA stability: effects of deletion of the intercistronic hairpin loop region of the Rhodobacter capsulatus puf operon.

It has been proposed that intercistronic stem and loop structures located in the puf operon of the photosynthetic bacterium Rhodobacter capsulatus account for segmental differences in transcript stability and consequently, the differential expression of the B870 and reaction center (RC) proteins encoded by puf. We report here that deletion of these structures leads to a failure to detect as discrete fragments the B870‐encoding 0.49 kb and 0.50 kb mRNA segments located upstream from the site of the hairpins. The absence of these stable transcript fragments is associated with altered stoichiometry of the B870 and RC pigment‐protein complexes in the bacterial intracytoplasmic membrane and a decreased rate of cell growth under photosynthetic conditions. These results support the view that the hairpin loop structures of the puf intercistronic region function in vivo to impede exoribonucleolytic degradation of upstream mRNA and establish that segmental variations in mRNA stability have a biologically important role in regulating the expression of puf operon genes.

The destabilizing elements in the coding region of c-fos mRNA are recognized as RNA.

The protein-coding region of the c-fos proto-oncogene transcript contains elements that direct the rapid deadenylation and decay of this mRNA in mammalian cells. The function of these coding region instability determinants requires movement of ribosomes across mRNAs containing them. Three types of mechanisms could account for this translational requirement. Two of these possibilities, (i) that rapid mRNA decay might be mediated by the nascent polypeptide chain and (ii) that it might result from an unusual codon usage, have experimental precedent. Here, we present evidence that the destabilizing elements in the c-fos coding region are not recognized in either of these two ways. Instead, the ability of the c-fos coding region to function as a potent mRNA destabilizer when translated in the +1 reading frame indicates that the signals for rapid deadenylation and decay reside in the sequence or structure of the RNA comprising this c-fos domain.

Consequences of RNase E scarcity in Escherichia coli

The endoribonuclease RNase E plays an important role in RNA processing and degradation in Escherichia coli. The construction of an E. coli strain in which the cellular concentration of RNase E can be precisely controlled has made it possible to examine and quantify the effect of RNase E scarcity on RNA decay, gene regulation and cell growth. These studies show that RNase E participates in a step in the degradation of its RNA substrates that is partially or fully rate‐determining. Our data also indicate that E. coli growth requires a cellular RNase E concentration at least 10–20% of normal and that the feedback mecha‐nism that limits overproduction of RNase E is also able to increase its synthesis when its concentration drops below normal. The magnitude of the in‐crease in RNA longevity under conditions of RNase E scarcity may be limited by an alternative pathway for RNA degradation. Additional experiments show that RNase E is a stable protein in E. coli. No other E. coli gene product, when either mutated or cloned on a multicopy plasmid, seems to be capable of compensating for an inadequate supply of this essential protein.