Enhanced Loading of Functional miRNA Cargo via pH Gradient Modification of Extracellular Vesicles.

Based on their identification as physiological nucleic acid carriers in humans and other organisms, extracellular vesicles (EVs) have been explored as therapeutic delivery vehicles for DNA, RNA, and other cargo. However, efficient loading and functional delivery of nucleic acids remain a challenge, largely because of potential sources of degradation and aggregation. Here, we report that protonation of EVs to generate a pH gradient across EV membranes can be utilized to enhance vesicle loading of nucleic acid cargo, specifically microRNA (miRNA), small interfering RNA (siRNA), and single-stranded DNA (ssDNA). The loading process did not impair cellular uptake of EVs, nor did it promote any significant EV-induced toxicity response in mice. Cargo functionality was verified by loading HEK293T EVs with either pro- or anti-inflammatory miRNAs and observing the effective regulation of corresponding cellular cytokine levels. Critically, this loading increase is comparable with what can be accomplished by methods such as sonication and electroporation, and is achievable without the introduction of energy associated with these methods that can potentially damage labile nucleic acid cargo.

Lack of tRNA-i6A modification causes mitochondrial-like metabolic deficiency in S. pombe by limiting activity of cytosolic tRNATyr, not mito-tRNA.

tRNA-isopentenyl transferases (IPTases) are highly conserved enzymes that form isopentenyl-N(6)-A37 (i6A37) on subsets of tRNAs, enhancing their translation activity. Nuclear-encoded IPTases modify select cytosolic (cy-) and mitochondrial (mt-) tRNAs. Mutation in human IPTase, TRIT1, causes disease phenotypes characteristic of mitochondrial translation deficiency due to mt-tRNA dysfunction. Deletion of the Schizosaccharomyces pombe IPTase (tit1-Δ) causes slow growth in glycerol, as well as in rapamycin, an inhibitor of TOR kinase that maintains metabolic homeostasis. Schizosaccharomyces pombe IPTase modifies three different cy-tRNAs(Ser) as well as cy-tRNA(Tyr), cy-tRNA(Trp), and mt-tRNA(Trp). We show that lower ATP levels in tit1-Δ relative to tit1(+) cells are also more decreased by an inhibitor of oxidative phosphorylation, indicative of mitochondrial dysfunction. Here we asked if the tit1-Δ phenotypes are due to hypomodification of cy-tRNA or mt-tRNA. A cytosol-specific IPTase that modifies cy-tRNA, but not mt-tRNA, fully rescues the tit1-Δ phenotypes. Moreover, overexpression of cy-tRNAs also rescues the phenotypes, and cy-tRNA(Tyr) alone substantially does so. Bioinformatics indicate that cy-tRNA(Tyr) is most limiting for codon demand in tit1-Δ cells and that the cytosolic mRNAs most loaded with Tyr codons encode carbon metabolilizing enzymes, many of which are known to localize to mitochondria. Thus, S. pombe i6A37 hypomodification-associated metabolic deficiency results from hypoactivity of cy-tRNA, mostly tRNA(Tyr), and unlike human TRIT1-deficiency does not impair mitochondrial translation due to mt-tRNA hypomodification. We discuss species-specific aspects of i6A37. Specifically relevant to mitochondria, we show that its hypermodified version, ms2i6A37 (2-methylthiolated), which occurs on certain mammalian mt-tRNAs (but not cy-tRNAs), is not found in yeast.

RNA Polymerase III Output Is Functionally Linked to tRNA Dimethyl-G26 Modification.

Control of the differential abundance or activity of tRNAs can be important determinants of gene regulation. RNA polymerase (RNAP) III synthesizes all tRNAs in eukaryotes and it derepression is associated with cancer. Maf1 is a conserved general repressor of RNAP III under the control of the target of rapamycin (TOR) that acts to integrate transcriptional output and protein synthetic demand toward metabolic economy. Studies in budding yeast have indicated that the global tRNA gene activation that occurs with derepression of RNAP III via maf1-deletion is accompanied by a paradoxical loss of tRNA-mediated nonsense suppressor activity, manifested as an antisuppression phenotype, by an unknown mechanism. We show that maf1-antisuppression also occurs in the fission yeast S. pombe amidst general activation of RNAP III. We used tRNA-HydroSeq to document that little changes occurred in the relative levels of different tRNAs in maf1Δ cells. By contrast, the efficiency of N2,N2-dimethyl G26 (m(2)2G26) modification on certain tRNAs was decreased in response to maf1-deletion and associated with antisuppression, and was validated by other methods. Over-expression of Trm1, which produces m(2)2G26, reversed maf1-antisuppression. A model that emerges is that competition by increased tRNA levels in maf1Δ cells leads to m(2)2G26 hypomodification due to limiting Trm1, reducing the activity of suppressor-tRNASerUCA and accounting for antisuppression. Consistent with this, we show that RNAP III mutations associated with hypomyelinating leukodystrophy decrease tRNA transcription, increase m(2)2G26 efficiency and reverse antisuppression. Extending this more broadly, we show that a decrease in tRNA synthesis by treatment with rapamycin leads to increased m(2)2G26 modification and that this response is conserved among highly divergent yeasts and human cells.

Engineered Multivalency Enhances Affibody-Based HER3 Inhibition and Downregulation in Cancer Cells.

The receptor tyrosine kinase HER3 has emerged as a therapeutic target in ovarian, prostate, breast, lung, and other cancers due to its ability to potently activate the PI3K/Akt pathway, especially via dimerization with HER2, as well as for its role in mediating drug resistance. Enhanced efficacy of HER3-targeted therapeutics would therefore benefit a wide range of patients. This study evaluated the potential of multivalent presentation, through protein engineering, to enhance the effectiveness of HER3-targeted affibodies as alternatives to monoclonal antibody therapeutics. Assessment of multivalent affibodies on a variety of cancer cell lines revealed their broad ability to improve inhibition of Neuregulin (NRG)-induced HER3 and Akt phosphorylation compared to monovalent analogues. Engineered multivalency also promoted enhanced cancer cell growth inhibition by affibodies as single agents and as part of combination therapy approaches. Mechanistic investigations revealed that engineered multivalency enhanced affibody-mediated HER3 downregulation in multiple cancer cell types. Overall, these results highlight the promise of engineered multivalency as a general strategy for enhanced efficacy of HER3-targeted therapeutics against a variety of cancers.

Defective i6A37 modification of mitochondrial and cytosolic tRNAs results from pathogenic mutations in TRIT1 and its substrate tRNA.

Identifying the genetic basis for mitochondrial diseases is technically challenging given the size of the mitochondrial proteome and the heterogeneity of disease presentations. Using next-generation exome sequencing, we identified in a patient with severe combined mitochondrial respiratory chain defects and corresponding perturbation in mitochondrial protein synthesis, a homozygous p.Arg323Gln mutation in TRIT1. This gene encodes human tRNA isopentenyltransferase, which is responsible for i6A37 modification of the anticodon loops of a small subset of cytosolic and mitochondrial tRNAs. Deficiency of i6A37 was previously shown in yeast to decrease translational efficiency and fidelity in a codon-specific manner. Modelling of the p.Arg323Gln mutation on the co-crystal structure of the homologous yeast isopentenyltransferase bound to a substrate tRNA, indicates that it is one of a series of adjacent basic side chains that interact with the tRNA backbone of the anticodon stem, somewhat removed from the catalytic center. We show that patient cells bearing the p.Arg323Gln TRIT1 mutation are severely deficient in i6A37 in both cytosolic and mitochondrial tRNAs. Complete complementation of the i6A37 deficiency of both cytosolic and mitochondrial tRNAs was achieved by transduction of patient fibroblasts with wild-type TRIT1. Moreover, we show that a previously-reported pathogenic m.7480A>G mt-tRNASer(UCN) mutation in the anticodon loop sequence A36A37A38 recognised by TRIT1 causes a loss of i6A37 modification. These data demonstrate that deficiencies of i6A37 tRNA modification should be considered a potential mechanism of human disease caused by both nuclear gene and mitochondrial DNA mutations while providing insight into the structure and function of TRIT1 in the modification of cytosolic and mitochondrial tRNAs.

Exogenous DNA Loading into Extracellular Vesicles via Electroporation is Size-Dependent and Enables Limited Gene Delivery.

Extracellular vesicles (EVs) hold immense promise for utilization as biotherapeutics and drug delivery vehicles due to their nature as biological nanoparticles that facilitate intercellular molecular transport. Specifically, EVs have been identified as natural carriers of nucleic acids, sparking interest in their use for gene therapy and RNA interference applications. So far, small RNAs (siRNA and miRNA) have been successfully loaded into EVs for a variety of delivery applications, but the potential use of EVs for DNA delivery has scarcely been explored. Here, we report that exogenous linear DNA can be associated with EVs via electroporation in quantities sufficient to yield an average of hundreds of DNA molecules per vesicle. We determined that loading efficiency and capacity of DNA in EVs is dependent on DNA size, with linear DNA molecules less than 1000 bp in length being more efficiently associated with EVs compared to larger linear DNAs and plasmid DNAs using this approach. We further showed that EV size is also determinant with regard to DNA loading, as larger microvesicles encapsulated more linear and plasmid DNA than smaller, exosome-like EVs. Additionally, we confirmed the ability of EVs to transfer foreign DNA loaded via electroporation into recipient cells, although functional gene delivery was not observed. These results establish critical parameters that inform the potential use of EVs for gene therapy and, in agreement with other recent results, suggest that substantial barriers must be overcome to establish EVs as broadly applicable DNA delivery vehicles.

Plasticity and diversity of tRNA anticodon determinants of substrate recognition by eukaryotic A37 isopentenyltransferases.

The N(6)-(isopentenyl)adenosine (i(6)A) modification of some tRNAs at position A37 is found in all kingdoms and facilitates codon-specific mRNA decoding, but occurs in different subsets of tRNAs in different species. Here we examine yeasts' tRNA isopentenyltransferases (i.e., dimethylallyltransferase, DMATase, members of the Δ(2)-isopentenylpyrophosphate transferase, IPPT superfamily) encoded by tit1(+) in Schizosaccharomyces pombe and MOD5 in Saccharomyces cerevisiae, whose homologs are Escherichia coli miaA, the human tumor suppressor TRIT1, and the Caenorhabditis elegans life-span gene product GRO-1. A major determinant of miaA activity is known to be the single-stranded tRNA sequence, A36A37A38, in a stem-loop. tRNA(Trp)(CCA) from either yeast is a Tit1p substrate, but neither is a Mod5p substrate despite the presence of A36A37A38. We show that Tit1p accommodates a broader range of substrates than Mod5p. tRNA(Trp)(CCA) is distinct from Mod5p substrates, which we sort into two classes based on the presence of G at position 34 and other elements. A single substitution of C34 to G converts tRNA(Trp)(CCA) to a Mod5p substrate in vitro and in vivo, consistent with amino acid contacts to G34 in existing Mod5p-tRNA(Cys)(GCA) crystal structures. Mutation of Mod5p in its G34 recognition loop region debilitates it differentially for its G34 (class I) substrates. Multiple alignments reveal that the G34 recognition loop sequence of Mod5p differs significantly from Tit1p, which more resembles human TRIT1 and other DMATases. We show that TRIT1 can also modify tRNA(Trp)(CCA) consistent with broad recognition similar to Tit1p. This study illustrates previously unappreciated molecular plasticity and biological diversity of the tRNA-isopentenyltransferase system of eukaryotes.

Selection of peptides targeting helix 31 of bacterial 16S ribosomal RNA by screening M13 phage-display libraries.

Ribosomal RNA is the catalytic portion of ribosomes, and undergoes a variety of conformational changes during translation. Structural changes in ribosomal RNA can be facilitated by the presence of modified nucleotides. Helix 31 of bacterial 16S ribosomal RNA harbors two modified nucleotides, m²G966 and m5C967, that are highly conserved among bacteria, though the degree and nature of the modifications in this region are different in eukaryotes. Contacts between helix 31 and the P-site tRNA, initiation factors, and ribosomal proteins highlight the importance of this region in translation. In this work, a heptapeptide M13 phage-display library was screened for ligands that target the wild-type, naturally modified bacterial helix 31. Several peptides, including TYLPWPA, CVRPFAL, TLWDLIP, FVRPFPL, ATPLWLK, and DIRTQRE, were found to be prevalent after several rounds of screening. Several of the peptides exhibited moderate affinity (in the high nM to low µM range) to modified helix 31 in biophysical assays, including surface plasmon resonance (SPR), and were also shown to bind 30S ribosomal subunits. These peptides also inhibited protein synthesis in cell-free translation assays.

Venezuelan equine encephalitis vaccine with rearranged genome resists reversion and protects non-human primates from viremia after aerosol challenge.

Live-attenuated V4020 vaccine for Venezuelan equine encephalitis virus (VEEV) containing attenuating rearrangement of the virus structural genes was evaluated in a non-human primate model for immunogenicity and protective efficacy against aerosol challenge with wild-type VEEV. The genomic RNA of V4020 vaccine virus was encoded in the pMG4020 plasmid under control of the CMV promoter and contained the capsid gene downstream from the glycoprotein genes. It also included attenuating mutations from the VEE TC83 vaccine, with E2-120Arg substitution genetically engineered to prevent reversion mutations. The population of V4020 vaccine virus derived from pMG4020-transfected Vero cells was characterized by next generation sequencing (NGS) and indicated no detectable genetic reversions. Cynomolgus macaques were vaccinated with V4020 vaccine virus. After one or two vaccinations including by intramuscular route, high levels of virus-neutralizing antibodies were confirmed with no viremia or apparent adverse reactions to vaccinations. The protective effect of vaccination was evaluated using an aerosol challenge with VEEV. After challenge, macaques had no detectable viremia, demonstrating a protective effect of vaccination with live V4020 VEEV vaccine.

Production of Extracellular Vesicles Loaded with Therapeutic Cargo.

Extracellular vesicles (EVs) are biological nanoparticles comprising exosomes, microvesicles, and other heterogeneous nanoscopic vesicle populations that are produced by most cell types. In addition to their putative roles as critical mediators of intercellular communication, EVs have begun to be harnessed as drug delivery vehicles, with early evidence indicating they may have significant advantages over synthetic nanoparticle delivery systems for particular applications. Targeted delivery of EV-encapsulated cargo has already been realized and may have broad applicability; however, methods for producing and purifying EVs and loading them with therapeutic molecules have yet to be standardized. In this chapter, we outline steps for EV isolation and characterization and compare current methods for active and passive loading of EVs with payloads of short interfering RNA (siRNA) or small molecules, with the results revealing that active loading via electroporation increases loading efficiency of siRNA but not of Rhodamine B, a model for a small molecule drug, in HEK293T-derived EVs. The methods described here may inform future design of targeted delivery of nucleic acids or small molecules via EVs.

Ethanol Induces Enhanced Vascularization Bioactivity of Endothelial Cell-Derived Extracellular Vesicles via Regulation of MicroRNAs and Long Non-Coding RNAs.

Extracellular vesicles (EVs), such as exosomes, have been identified as regulators of vascular remodeling and have promise as therapeutics for vascularization applications. Towards development of EVs as therapeutics, it has been demonstrated that physiological stimuli of angiogenic phenotypes in EV-producing cells can enhance the potency of EVs for vascularization. The goal of this study was to assess whether ethanol, which induces angiogenic phenotypes in endothelial cells, could be employed to enhance endothelial-derived EV vascularization bioactivity. The results indicate that ethanol conditioning of endothelial cells increases the ability of endothelial EVs to induce a pro-vascularization response. This response is due in part to increased CD34 expression in recipient endothelial cells that may result from downregulation of microRNA-106b in EVs isolated from ethanol-conditioned producer endothelial cells. Further, ethanol-induced upregulation of long non-coding RNAs (lncRNAs) HOTAIR and MALAT1 in endothelial EVs was observed to play a significant role in mediating pro-angiogenic effects of these vesicles. Overall, these studies validate ethanol conditioning as a method to enhance the bioactivity of endothelial EVs via regulation of EV-associated microRNAs (miRNAs) and, especially, lncRNAs. Further, the results suggest that alcohol consumption may activate endothelial EVs towards a pro-vascularization phenotype, which could have implications for alcohol-induced tumor angiogenesis.

Impact of cell culture parameters on production and vascularization bioactivity of mesenchymal stem cell-derived extracellular vesicles.

Mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs) have emerged as potential therapeutic agents for numerous applications. EVs offer potential advantages over cell-based therapies with regard to safety, stability and clearance profiles, however production and potency limitations must be addressed to enable eventual translation of EV-based approaches. Thus, we sought to examine the role of specific cell culture parameters on MSC EV production and bioactivity toward informing rational design parameters for scalable EV biomanufacturing. We report significantly reduced MSC EV vascularization bioactivity, as measured by an endothelial cell gap closure assay, with increasing passage in culture by trypsinization, especially beyond passage 4. We further show that increased frequency of EV collection yielded higher numbers of EVs from the same initial number of MSCs over a 24 hr period. Finally, we demonstrate that decreased cell seeding density in culture flasks resulted in increased production of EVs per cell in MSCs and other cell types. Overall, these studies highlight the need for careful consideration of the parameters of cell passage number and cell seeding density in the production of therapeutic EVs at laboratory scale and for rational design of large-scale EV biomanufacturing schemes.

Oncogene Knockdown via Active Loading of Small RNAs into Extracellular Vesicles by Sonication.

Extracellular vesicles (EVs), including exosomes and microvesicles, have emerged as promising drug delivery vehicles for small RNAs (siRNA and miRNA) due to their natural role in intercellular RNA transport. However, the application of EVs for therapeutic RNA delivery may be limited by loading approaches that can induce cargo aggregation or degradation. Here, we report the use of sonication as a means to actively load functional small RNAs into EVs. Conditions under which EVs could be loaded with small RNAs with minimal detectable aggregation were identified, and EVs loaded with therapeutic siRNA via sonication were observed to be taken up by recipient cells and capable of target mRNA knockdown leading to reduced protein expression. This system was ultimately applied to reduce expression of HER2, an oncogenic receptor tyrosine kinase that critically mediates breast cancer development and progression, and could be extended to other therapeutic targets. These results define important parameters informing the application of sonication as a small RNA loading method for EVs and demonstrate the potential utility of this approach for versatile cancer therapy.

Emerging roles for extracellular vesicles in tissue engineering and regenerative medicine.

Extracellular vesicles (EVs)-comprising a heterogeneous population of cell-derived lipid vesicles including exosomes, microvesicles, and others-have recently emerged as both mediators of intercellular information transfer in numerous biological systems and vehicles for drug delivery. In both roles, EVs have immense potential to impact tissue engineering and regenerative medicine applications. For example, the therapeutic effects of several progenitor and stem cell-based therapies have been attributed primarily to EVs secreted by these cells, and EVs have been recently reported to play direct roles in injury-induced tissue regeneration processes in multiple physiological systems. In addition, EVs have been utilized for targeted drug delivery in regenerative applications and possess unique potential to be harnessed as patient-derived drug delivery vehicles for personalized medicine. This review discusses EVs in the context of tissue repair and regeneration, including their utilization as drug carriers and their crucial role in cell-based therapies. Furthermore, the article highlights the growing need for bioengineers to understand, consider, and ultimately design and specifically control the activity of EVs to maximize the efficacy of tissue engineering and regenerative therapies.

Human cells have a limited set of tRNA anticodon loop substrates of the tRNA isopentenyltransferase TRIT1 tumor suppressor.

Human TRIT1 is a tRNA isopentenyltransferase (IPTase) homologue of Escherichia coli MiaA, Saccharomyces cerevisiae Mod5, Schizosaccharomyces pombe Tit1, and Caenorhabditis elegans GRO-1 that adds isopentenyl groups to adenosine 37 (i6A37) of substrate tRNAs. Prior studies indicate that i6A37 increases translation fidelity and efficiency in codon-specific ways. TRIT1 is a tumor suppressor whose mutant alleles are associated with cancer progression. We report the systematic identification of i6A37-containing tRNAs in a higher eukaryote, performed using small interfering RNA knockdown and other methods to examine TRIT1 activity in HeLa cells. Although several potential substrates contained the IPTase recognition sequence A36A37A38 in the anticodon loop, only tRNA(Ser)AGA, tRNA(Ser)CGA, tRNA(Ser)UGA, and selenocysteine tRNA with UCA (tRNA([Ser]Sec)UCA) contained i6A37. This subset is a significantly more restricted than that for two distant yeasts (S. cerevisiae and S. pombe), the only other organisms comprehensively examined. Unlike the fully i6A37-modified tRNAs for Ser, tRNA([Ser]Sec)UCA is partially (∼40%) modified. Exogenous selenium and other treatments that decreased the i6A37 content of tRNA([Ser]Sec)UCA led to increased levels of the tRNA([Ser]Sec)UCA. Of the human mitochondrion (mt)-encoded tRNAs with A36A37A38, only mt tRNAs tRNA(Ser)UGA and tRNA(Trp)UCA contained detectable i6A37. Moreover, while tRNA(Ser) levels were unaffected by TRIT1 knockdown, the tRNA([Ser]Sec)UCA level was increased and the mt tRNA(Ser)UGA level was decreased, suggesting that TRIT1 may control the levels of some tRNAs as well as their specific activity.

Lack of tRNA modification isopentenyl-A37 alters mRNA decoding and causes metabolic deficiencies in fission yeast.

tRNA isopentenyltransferases (Tit1) modify tRNA position 37, adjacent to the anticodon, to N6-isopentenyladenosine (i6A37) in all cells, yet the tRNA subsets selected for modification vary among species, and their relevance to phenotypes is unknown. We examined the function of i6A37 in Schizosaccharomyces pombe tit1+ and tit1-Δ cells by using a ß-galactosidase codon-swap reporter whose catalytic activity is sensitive to accurate decoding of codon 503. i6A37 increased the activity of tRNACys at a cognate codon and that of tRNATyr at a near-cognate codon, suggesting that i6A37 promotes decoding activity generally and increases fidelity at cognate codons while decreasing fidelity at noncognate codons. S. pombe cells lacking tit1+ exhibit slow growth in glycerol or rapamycin. While existing data link wobble base U34 modifications to translation of functionally related mRNAs, whether this might extend to the anticodon-adjacent position 37 was unknown. Indeed, we found a biased presence of i6A37-cognate codons in high-abundance mRNAs for ribosome subunits and energy metabolism, congruent with the observed phenotypes and the idea that i6A37 promotes translational efficiency. Polysome profiles confirmed the decreased translational efficiency of mRNAs in tit1-Δ cells. Because subsets of i6A37-tRNAs differ among species, as do their cognate codon-sensitive mRNAs, these genomic variables may underlie associated phenotypic differences.

3' processing of eukaryotic precursor tRNAs.

Biogenesis of eukaryotic tRNAs requires transcription by RNA polymerase III and subsequent processing. 5' processing of precursor tRNA occurs by a single mechanism, cleavage by RNase P, and usually occurs before 3' processing although some conditions allow observation of the 3'-first pathway. 3' processing is relatively complex and is the focus of this review. Precursor RNA 3'-end formation begins with pol III termination generating a variable length 3'-oligo(U) tract that represents an underappreciated and previously unreviewed determinant of processing. Evidence that the pol III-intrinsic 3'exonuclease activity mediated by Rpc11p affects 3'oligo(U) length is reviewed. In addition to multiple 3' nucleases, precursor tRNA(pre-tRNA) processing involves La and Lsm, distinct oligo(U)-binding proteins with proposed chaperone activities. 3' processing is performed by the endonuclease RNase Z or the exonuclease Rex1p (possibly others) along alternate pathways conditional on La. We review a Schizosaccharomyces pombe tRNA reporter system that has been used to distinguish two chaperone activities of La protein to its two conserved RNA binding motifs. Pre-tRNAs with structural impairments are degraded by a nuclear surveillance system that mediates polyadenylation by the TRAMP complex followed by 3'-digestion by the nuclear exosome which appears to compete with 3' processing. We also try to reconcile limited data on pre-tRNA processing and Lsm proteins which largely affect precursors but not mature tRNAs.A pathway is proposed in which 3' oligo(U) length is a primary determinant of La binding with subsequent steps distinguished by 3'-endo versus exo nucleases,chaperone activities, and nuclear surveillance.

Combined Approaches to Site-Specific Modification of RNA.

Both natural and unnatural modifications in RNA are of interest to biologists and chemists. More than 100 different analogues of the four standard RNA nucleosides have been identified in nature. Unnatural modifications are useful for structure and mechanistic studies of RNA. This Review highlights chemical, enzymatic, and combined (semisynthesis) approaches to generate site specifically modified RNAs. The availability of these methods for site-specific modifications of RNAs of all sizes is important in order to study the relationships between RNA chemical composition, structure, and function.

Identification and role of functionally important motifs in the 970 loop of Escherichia coli 16S ribosomal RNA.

The 970 loop (helix 31) of Escherichia coli 16S ribosomal RNA contains two modified nucleotides, m(2)G966 and m(5)C967. Positions A964, A969, and C970 are conserved among the Bacteria, Archaea, and Eukarya. The nucleotides present at positions 965, 966, 967, 968, and 971, however, are only conserved and unique within each domain. All organisms contain a modified nucleoside at position 966, but the type of the modification is domain specific. Biochemical and structure studies have placed this loop near the P site and have shown it to be involved in the decoding process and in binding the antibiotic tetracycline. To identify the functional components of this ribosomal RNA hairpin, the eight nucleotides of the 970 loop of helix 31 were subjected to saturation mutagenesis and 107 unique functional mutants were isolated and analyzed. Nonrandom nucleotide distributions were observed at each mutated position among the functional isolates. Nucleotide identity at positions 966 and 969 significantly affects ribosome function. Ribosomes with single mutations of m(2)G966 or m(5)C967 produce more protein in vivo than do wild-type ribosomes. Overexpression of initiation factor 3 specifically restored wild-type levels of protein synthesis to the 966 and 967 mutants, suggesting that modification of these residues is important for initiation factor 3 binding and for the proper initiation of protein synthesis.