Nuclear translation or nuclear peptidyl transferase?

Abstract

It is widely accepted that protein synthesis occurs in the cytoplasms of eukaryotic cells, but some investigators believe that it also occurs in the nucleus. In spite of experiments performed in several labs over many years, the issue of nuclear translation remains unresolved. Advocates assert that it would serve as an economical and convenient way to explain how cells monitor the quality of newly made mRNAs or ribosomes. Skeptics argue that regardless of its esthetic appeal, compelling evidence for nuclear translation has been absent.^1-3 The key question—also central to the debate more than 30 years ago—is whether alleged nuclear translation can be proven to represent “genuine polypeptide synthesis that is a function of the nuclear compartment”⁴.

Keywords: :

• nascent ribosomes
• nuclear translation
• peptidyl transferase
• puromycin

Recently, David et al.⁵ published evidence that they and others⁶ interpret as strongly favoring the idea that protein synthesis does occur in nuclei. David et al. devised a way to analyze the intracellular location of peptide synthesis in vivo that is based on visualization of nascent polypeptides whose carboxyl termini are tagged with puromycin (PMY). In the presence of the protein synthesis inhibitors cycloheximide or emetine, which block ribosome translocation, PMY-linked polypeptides remain on ribosomes and thus mark functioning ribosomes with a PMY-tag. The locations of these ribosomes within cells are then determined by fluorescence microscopy using PMY-specific antibodies. Using this method David et al.⁵ detected immobilized PMY in both the nucleus and cytoplasm of treated cells and these signals partially overlapped with the immunofluorescence signals from the acidic P proteins of the large (60S) ribosomal subunit. Importantly, they demonstrated that inhibitors of the peptidyl transferase activity of 60S subunits⁷ prevented generation of the PMY-signals in both the cytoplasm and nucleus. Because translating ribosomes are unlikely to be rapidly imported from the cytoplasm, the authors concluded that the nuclear PMY-signals, which are concentrated in nucleoli, resulted from protein synthesis that had occurred within the nucleus.

In other experiments, the cells were first infected with a variety of RNA viruses that interfere with host cell protein synthesis and mRNA metabolism. In most cases, such cells exhibited no detectable nuclear PMY signal, possibly indicating that nuclear translation requires newly made nuclear mRNA. However, the lack of signal might also have resulted from virus-induced inhibition of new ribosome production, which would have led to the absence of peptidyl transferase activity of maturing ribosome subunits in the nuclei.

While nuclear translation could explain these results, the critical problem with these experiments is the lack of characterization of the PMY-products that generate the nuclear signals; the authors indicate that they are planning to undertake this analysis. Although ribosome-bound, heterogeneous high-molecular-weight peptidyl-PMY products are detected in total cell extracts, these extracts are primarily cytoplasmic in origin so it is unclear whether any of these PMY-peptides were generated in the nucleus. Instead, it is posible that the nuclear signals may be derived from other, non-polypeptide products. For example, peptidyl transferase activity associated with the nascent 60S ribosomal subunits assembling in nucleoli might catalyze attachment of PMY to pre-existing nuclear material or to amino acids donated by aminoacyl-tRNAs, which are present in nuclei.⁸ Recently, others have raised concerns about the use of puromycin tagging to detect intracellular sites of peptide bond formation.^9,10

Several features of the products responsible for the nuclear PMY signals remain unexplained. In some figures these signals do not overlap well with signals of acidic ribosomal P proteins, and their persistence for at least 6 or 7 h seems surprisingly long for prematurely-terminated polypeptides. Unfortunately, no comparative data are presented for the longevity of the cytoplasmic PMY-polypeptides.

In principle, it would be possible to first purify nucleoli, which contain much of the nuclear signal, and then analyze the PMY-containing products. Linkage of PMY to a heterogeneous mixture of nascent polypeptides would indicate that they were made by a bona fide mRNA-templated translation process, whereas incorporation of PMY into more discrete size products would indicate a non-templated condensation mediated by immature 60S ribosomal subunits. Although condensation of PMY with an amino acid obtained from aminoacyl-tRNA would indeed constitute nuclear peptide bond formation it would differ from template-directed protein synthesis that is strictly dependent on active, mature 80S ribosomes.¹¹ In this regard it is tempting to speculate that nuclear peptide bond formation – as opposed to nuclear translation – normally might serve to proofread the newly generated peptidyl-transferase center of maturing, nascent 60S ribosomal subunits, as discussed previously.¹²

Disregarding the uncertainty about the nature of nuclear PMY products, the paper by David et al. provides an attractive new approach to assess protein synthesis in living cells, and it may well offer the long sought-after method to clearly establish if translation actually occurs within nuclei. However, until the nuclear PMY-products can be unambiguously identified as tagged polypeptides synthesized on intra-nuclear 80S ribosomes there is still reason to be skeptical about the existence of nuclear translation.