Beyond rRNA and snRNA: tRNA as a 2'-O-methylation target for nucleolar and Cajal body box C/D RNPs.

Box C/D small nucleolar RNAs (snoRNAs) and small Cajal body (CB) RNAs (scaRNAs) form ribonucleoprotein (RNP) complexes to mediate 2'-O-methylation of rRNAs and small nuclear RNAs (snRNAs), respectively. The site of methylation is determined by antisense elements in the box C/D RNAs that are complementary to sequences in target RNAs. However, numerous box C/D RNAs in mammalian cells lack antisense elements to rRNAs or snRNAs; thus, their targets remain unknown. In this issue of Genes & Development, Vitali and Kiss (pp. 741-746) demonstrate that "orphan" nucleolar box C/D snoRNA SNORD97 and CB box C/D scaRNA SCARNA97 contain antisense elements that target the wobble cytidine at position 34 of human elongator tRNAMet(CAT) for 2'-O-methylation (C34m). C34m is jointly mediated by SNORD97 and SCARNA97 despite their apparently different intranuclear locations. Furthermore, the investigators demonstrate that C34m prohibits site-specific cleavage of tRNAMet (CAT) into tRNA fragments (tRFs) by the stress-responsive endoribonuclease angiogenin, thereby uncovering a role for SNORD97 and SCARNA97 in the biogenesis of tRFs, which modulate a diverse set of cellular functions in human health and disease.

A novel assay provides insight into tRNAPhe retrograde nuclear import and re-export in S. cerevisiae.

In eukaryotes, tRNAs are transcribed in the nucleus and subsequently exported to the cytoplasm where they serve as essential adaptor molecules in translation. However, tRNAs can be returned to the nucleus by the evolutionarily conserved process called tRNA retrograde nuclear import, before relocalization back to the cytoplasm via a nuclear re-export step. Several important functions of these latter two trafficking events have been identified, yet the pathways are largely unknown. Therefore, we developed an assay in Saccharomyces cerevisiae to identify proteins mediating tRNA retrograde nuclear import and re-export using the unique wybutosine modification of mature tRNAPhe. Our hydrochloric acid/aniline assay revealed that the karyopherin Mtr10 mediates retrograde import of tRNAPhe, constitutively and in response to amino acid deprivation, whereas the Hsp70 protein Ssa2 mediates import specifically in the latter. Furthermore, tRNAPhe is re-exported by Crm1 and Mex67, but not by the canonical tRNA exporters Los1 or Msn5. These findings indicate that the re-export process occurs in a tRNA family-specific manner. Together, this assay provides insights into the pathways for tRNAPhe retrograde import and re-export and is a tool that can be used on a genome-wide level to identify additional gene products involved in these tRNA trafficking events.

A Simple Method for the Detection of Wybutosine-Modified tRNAPheGAA as a Readout of Retrograde tRNA Nuclear Import and Re-export: HCl/Aniline Cleavage and Nonradioactive Northern Hybridization.

tRNAs are highly mobile molecules that are trafficked back and forth between the nucleus and cytoplasm by several proteins. However, characterization of the movement of tRNAs and the proteins mediating these movements can be difficult. Here, we describe an easy and cost-effective assay to discover genes that are involved in two specific tRNA trafficking events, retrograde nuclear import and nuclear re-export for yeast, Saccharomyces cerevisiae. This assay, referred to as the hydrochloric acid (HCl)/aniline assay, identifies the presence or absence of a unique modification on tRNAPheGAA called wybutosine (yW) that requires mature, spliced tRNAPheGAA to undergo retrograde nuclear import and subsequent nuclear re-export for its addition. Therefore, the presence/absence of yW-modified tRNAPheGAA serves as a readout of retrograde nuclear import and nuclear re-export. This simple assay can be used to determine the role of any gene product in these previously elusive tRNA trafficking events.

tRNA Processing and Subcellular Trafficking Proteins Multitask in Pathways for Other RNAs.

This article focuses upon gene products that are involved in tRNA biology, with particular emphasis upon post-transcriptional RNA processing and nuclear-cytoplasmic subcellular trafficking. Rather than analyzing these proteins solely from a tRNA perspective, we explore the many overlapping functions of the processing enzymes and proteins involved in subcellular traffic. Remarkably, there are numerous examples of conserved gene products and RNP complexes involved in tRNA biology that multitask in a similar fashion in the production and/or subcellular trafficking of other RNAs, including small structured RNAs such as snRNA, snoRNA, 5S RNA, telomerase RNA, and SRP RNA as well as larger unstructured RNAs such as mRNAs and RNA-protein complexes such as ribosomes. Here, we provide examples of steps in tRNA biology that are shared with other RNAs including those catalyzed by enzymes functioning in 5' end-processing, pseudoU nucleoside modification, and intron splicing as well as steps regulated by proteins functioning in subcellular trafficking. Such multitasking highlights the clever mechanisms cells employ for maximizing their genomes.

tRNA dynamics between the nucleus, cytoplasm and mitochondrial surface: Location, location, location.

Although tRNAs participate in the essential function of protein translation in the cytoplasm, tRNA transcription and numerous processing steps occur in the nucleus. This subcellular separation between tRNA biogenesis and function requires that tRNAs be efficiently delivered to the cytoplasm in a step termed "primary tRNA nuclear export". Surprisingly, tRNA nuclear-cytoplasmic traffic is not unidirectional, but, rather, movement is bidirectional. Cytoplasmic tRNAs are imported back to the nucleus by the "tRNA retrograde nuclear import" step which is conserved from budding yeast to vertebrate cells and has been hijacked by viruses, such as HIV, for nuclear import of the viral reverse transcription complex in human cells. Under appropriate environmental conditions cytoplasmic tRNAs that have been imported into the nucleus return to the cytoplasm via the 3rd nuclear-cytoplasmic shuttling step termed "tRNA nuclear re-export", that again is conserved from budding yeast to vertebrate cells. We describe the 3 steps of tRNA nuclear-cytoplasmic movements and their regulation. There are multiple tRNA nuclear export and import pathways. The different tRNA nuclear exporters appear to possess substrate specificity leading to the tantalizing possibility that the cellular proteome may be regulated at the level of tRNA nuclear export. Moreover, in some organisms, such as budding yeast, the pre-tRNA splicing heterotetrameric endonuclease (SEN), which removes introns from pre-tRNAs, resides on the cytoplasmic surface of the mitochondria. Therefore, we also describe the localization of the SEN complex to mitochondria and splicing of pre-tRNA on mitochondria, which occurs prior to the participation of tRNAs in protein translation. This article is part of a Special Issue entitled: SI: Regulation of tRNA synthesis and modification in physiological conditions and disease edited by Dr. Boguta Magdalena.