RNA Metabolism in Cell Biology, Growth, and Development

We study the biology and relevance to human health of mRNAs and tRNAs, the principal molecular messengers and adapter agents used to decipher and express our genetic code. The biogenesis, processing, modification, and decay of tRNAs and mRNAs, and isoforms and fragments thereof, as well as some of their specific interacting proteins that contribute to cell proliferation, growth, and development during health and disease, are the topics of our experimental investigations and other studies. Some studies focus on the La protein, which interacts with nuclear precursor tRNAs and the few other newly transcribed short transcripts synthesized by RNA polymerase III (Pol III), which are matured to small noncoding (nc) RNAs of diverse critical functions. The La protein is a unique chaperone for Pol III transcripts. It binds to and protects their shared 3′-oligo(U) motifs from exonucleolytic digestion and decay, can assist proper folding, and for many, retains them in the nucleus to acquire specific modifications and undergo specific RNA–processing steps prior to export into the cytoplasm. Surprisingly, accumulating evidence suggests that failure to produce properly matured Pol III ncRNAs can contribute to disease states independent of the requirement for normal processes, but also because they can activate cellular innate immune response and other surveillance pathways [reviewed in Reference 5]. Other studies focus on the La–related proteins (LARPs) 1 and 4, which share similar activities for RNA 3′ end binding and protection from exonucleolytic digestion, but for cytoplasmic mRNAs rather than for nuclear precursor ncRNAs. Related to mature functional tRNAs and their role in mRNA translation, we focus on the tRNA anticodon-loop modification enzyme known as tRNA-isopentenyltransferase-1 (TRIT1). The La protein is also referred to as "La antigen" for its long-known involvement as a target of auto-antibodies in patients with chronic inflammatory autoimmune disorders such as systemic lupus erythematosus (SLE), Sjögren’s syndrome (SS), and neonatal lupus. The official name for the gene encoding La protein is SSB (Sjögren’s syndrome antigen-B).

tRNAs are produced at over 10-fold higher molar equivalents than ribosomes during cellular proliferation. More than 500 unique human tRNA gene loci comprise the largest subset of genes actively transcribed by Pol III, followed by 15–30 nearly identical copies of 5S ribosomal RNA genes and several additional unique “single copy” ncRNA genes. The La protein serves to promote overall proper tRNA maturation. Many diseases are attributable to defective tRNA biogenesis and the consequent failure to support mRNA translation. Related to surveillance, Pol III itself acts directly in a dedicated innate immune activation pathway separate from its nuclear transcription role. In addition, a few Pol III–transcribed ncRNAs appear to have also been co-opted by the innate immune system [Reference 5]. Recent findings in our lab extend this potential to Pol III transcripts of a minority subset of (human) tRNA genes that can activate innate immune and inflammatory pathways. Further, this work indicates that the La protein can suppress the innate immune and inflammatory activity of these tRNA gene transcripts in human cells [Kessler et al, under revision].

Pol III synthesizes high levels of tRNA by a conserved process of transcription termination–associated re-initiation, relevant to development and cancer. Termination occurs within a short tract of T residues in the non-template (NT) DNA strand at the ends of Pol III–transcribed genes. The nascent transcripts bear a copy of this terminator, U(n)U-3′OH, a recognition motif for the nuclear La protein, which binds in a sequence- and length-dependent manner. Notably, the 3′U(n) length–dependence of La binding is usually shorter than the minimal T-length required for efficient Pol III termination, the latter of which is 6, 5, and 4 Ts for Saccharomyces cerevisiae, Schizosaccharomyces pombe, and humans, respectively. This suggests an La link with tRNA expression, a link that fits with human Pol III, which evolved a minimal 4T termination mechanism; the data suggest that this may direct some post-transcriptional events. Studies on development, structure, and its gene variants indicate that human Pol III evolved termination mechanisms to control gene-regulatory programs with greater intricacy.

The human La protein is a target of auto-antibodies in patients with chronic inflammatory diseases such as systemic lupus erythematosus, Sjögren’s syndrome (SS) (La is also known as SS antigen-B, SSB), and neonatal lupus. Although La is active in other immune pathways, evidence for its primary involvement in autoimmunity is lacking. As noted above, La binds to POL III transcripts in all cells and serves as a chaperone during their nuclear maturation. La not only protects pre–tRNAs from 3′ exonucleases, but also directs and temporally orders the first step in the 5′ processing by the RNase P pathway. We propose a role for La in a novel pathway, different from tRNA maturation. Our data suggest that La is a determinant for a subset of pre-tRNAs (genes) to enter an alternative pathway to specific activation of the type-I interferon (IFN) response. We are studying a dual-activity tRNA gene(s) for which La is a key determinant of whether the nascent 4T–terminated transcripts are directed to tRNA maturation or to an alternate pathway of innate immune activation. Accordingly, some dual-activity tRNA genes may serve as endogenous immune adjuvants or may contribute to autoimmunity. We are currently exploring these possibilities.

The rate of RNA production by Pol III was shown to affect the efficiency by which pre-tRNAs acquire the common m^2,2G26 modification, which increases tRNA activity. Disrupted tRNA biogenesis, attributable to gene variants, leads to developmental and other diseases, as well as to neurodegeneration and intellectual impairment. About 120 modifications occur on tRNAs, of which about 40 have been documented for human cytoplasmic (cy–) tRNAs and several others for human mitochondrial (mt-) tRNAs. Many tRNA–modification enzymes (TME) are multisubunit, and some composite modifications require several gene products. Thus, mutations in numerous TMEs cause disease.

Critical modifications to tRNA anticodon loops fine-tune base pairing for optimal decoding and wobble decoding of synonymous codons, which is an important component of a tunable system of high-fidelity cy–translation. It is important to note that the 61 sense codons for 20 amino acids (aa) are decoded by cy-tRNAs, which collectively carry only 45 anticodons, a feature of tRNA genes named anticodon-sparing, that is widely conserved, although the number and identity of the ‘missing’ anticodons differ among kingdoms, anti-correlated with wobble-base modifications. In humans, about 15 standard codons must be wobble-decoded by cy–tRNAs, whose activities are controlled by anticodon modifications. Thus, Pol III transcription of tRNA genes is only one level on which the translation of a range of cy–mRNAs with biased codon content can be regulated. While this constitutes a nuclear-cytoplasmic translation system with much potential for intricate regulation, the complexities of overlapping/redundant decoding activities also create potential for translation infidelity in cases of faulty modification or tRNA pool imbalance.

All TMEs are nuclear-encoded, and a minority subset modify both cy– and mt–tRNAs, with potential to synchronize translation in both compartments. Distinct decoding rules apply in mitochondria, in part because each mt–DNA encodes only one tRNA for each of 18 amino acids. Synonymous codons occupy mt–mRNAs, but are not distinguished by different tRNAs, as is the case for cy–mRNAs. For example, while four codons each for Ala, Gly, Pro, Thr, and Val are decoded by three cy–tRNAs, a single mt–tRNA for each must wobble-decode its four cognate codons. Furthermore, these mt-tRNAs use unmodified U34 as their wobble base, whereas cy–tRNA wobble bases are part of an elaborate modification system, especially for U34. Each mt–tRNA is critical; point mutations that impair mt–DNA translation are uniquely associated with oxidative-phosphorylation diseases, whereas most nuclear tRNA genes are buffered by multiple copies; a known exception is a unique, single-copy, brain-specific tRNA. Yet, some diseases caused by a mutation in a mt–tRNA exhibit a phenotype like that resulting from a nuclear gene mutation to a TME whose substrate is the mt–tRNA.

Numerous pathogenic alleles that encode defective subunits of Pol III cause hypomyelinating leukodystrophy (HLD), whose pathobiology is consistent with poor formation of axonal myelin sheaths rather than demyelination. Pol III produces several ncRNAs (non-coding RNAs) in addition to tRNAs. We suggest that another disruption mechanism of a tRNA biogenesis–CNS network is also possible. As TME defects often manifest as neurological, reflecting tissue reliance on mitochondria, and because cy– and mt–translation are synchronized, including by tRNA modifications and other factors, disruption of general tRNA homeostasis may impair mitochondrial function, with a pathophysiologic contribution to disease, including in developing oligodendrocytes.

mRNA levels are determined in significant part by levels of cognate tRNAs, from yeast to human. In addition, certain mRNAs are particularly sensitive to tRNA levels. The LARP4 mRNA has a tract of about 70 codons with a very poor match to cellular tRNA levels. Thus, unprogrammed changes in tRNA output (e.g., genetic mutation in POL III) could shift mRNA stabilities and translation efficiencies in unpredictable ways with uncertain outcomes.

Translation of LARP4 mRNA produces LARP4, which binds to poly(A) and to poly(A)–binding protein (PABP/PABPC1). LARP4 stabilizes mRNAs by opposing deadenylation of their poly(A) tails, substrates of Ccr4-Not deadenylase (a multi-protein complex that functions in gene expression in the nucleus, where it regulates transcription, and in the cytoplasm, where it associates with translating ribosomes and RNA–processing bodies). Potential for regulation is that Ccr4-Not monitors mRNA–ribosomes for codon–tRNA match. For ribosomal protein–encoding mRNAs stabilized by LARP4, this supports a working model in which LARP4 mRNA senses tRNA levels and relays this by producing LARP4 to regulate ribosome biogenesis, perhaps with LARP1.

It has been known for some time that human Pol III and associated factors are dysregulated in cancer. More recently, functionally related mRNAs favoring proliferative or differentiation states were shown to be biased in synonymous codons and in the cognate tRNAs differentially expressed in those cells. Strikingly, the first Pol III structures provide insight into activities specific to the higher eukaryotic 17-subunit complex. First, the cancer-associated RPC7a Pol III subunit paralog (encoded by POLR3G) appeared to interfere with binding of the Pol III–negative regulator and tumor suppressor MAF1, whereas the RPC7b (POLR3GL) paralog subunit is enriched in cells programmed for differentiation (limited proliferation). Yeast Pol III has only one homolog. The second example involves the most striking feature of higher eukaryote–specific Pol III: the multi-domain expansion of RPC5 and hypothesized associated higher eukaryote promoter-type specificity, and its link to termination-reinitiation recycling.

Mechanistic control of ncRNA genes appears to have evolved activities that extend human Pol III beyond the housekeeping activities of its yeast counterpart. This fits with a view of hPol III in self vs. non-self surveillance functions. Notable are Pol III Vault (Vt) ncRNAs (Vault is a hollow barrel-shaped ribonucleoprotein complex) with involvement in two activity types: innate immune surveillance, and differentiation vs. maintenance of undifferentiated states. Both Pol III Vt and snaR (small nuclear factor 90–associated RNA) ncRNAs are processed to miRNAs that exert downstream effects on mRNA profiles and/or differentiation/cancer.