Patrick Linder

DExD/H Box RNA Helicases: From Generic Motors to Specific Dissociation Functions

RNA helicases of the DEAD box and related DExD/H proteins form a very large superfamily of proteins conserved from bacteria and viruses to humans. They have seven to eight conserved motifs, the characteristics of which are used to subgroup members into individual families. They are associated with all processes involving RNA molecules, including transcription, editing, splicing, ribosome biogenesis, RNA export, translation, RNA turnover, and organelle gene expression. Analysis of the three-dimensional structures obtained through the crystallization of viral and cellular RNA helicases reveals a strong structural homology to DNA helicases. In this review, we discuss our current understanding of RNA helicases and their biological function.

DEAD-BOX PROTEINS: THE DRIVING FORCES BEHIND RNA METABOLISM

RNA helicases from the DEAD-box family are found in almost all organisms and have important roles in RNA metabolism. They are associated with many processes ranging from RNA synthesis to RNA degradation. DEAD-box proteins use the energy from ATP hydrolysis to rearrange inter- or intra-molecular RNA structures or dissociate RNA–protein complexes. Such dynamic rearrangements are fundamental for many, if not all, steps in the life of an RNA molecule. Recent biochemical, genetic and structural data shed light on how these proteins power the metabolism of RNA within a cell.

Unwinding RNA in Saccharomyces cerevisiae: DEAD-box proteins and related families

Members of the RNA-helicase family are defined by several evolutionary conserved motifs. They are found in all organisms - from bacteria to humans - and many viruses. The minimum number of RNA helicases present within a eukaryotic cell can be predicted from the complete sequence of the Saccharomyces cerevisiae genome. Recent progress in the functional analysis of various family members has given new insights into, and confirmed the significance of these proteins for, most cellular RNA metabolic processes.

D-E-A-D protein family of putative RNA helicases

RNA metabolism plays a central role in cell growth. It is essential to regulate RNA synthesis, processing, stability and degradation. Conformational changes in RNA are key elements in regulating cellular processes. Recently, an increasing number of putative RNA helicases from different organisms ranging from Escherichia coli to humans and viruses have been identified. They are Involved in diverse cellular functions such as RNA splicing, ribosome assembly, initiation of translation, spermatogenesis, embryogenesis, and cell growth and division. Based on sequence homologies these proteins were grouped in a family, the D‐E‐A‐D box protein family (D‐E‐A‐D = Asp‐Glu‐Ala‐Asp). Some of the better characterized members have been shown to possess ATP‐binding and hydrolysing activities as well as ATP‐dependent RNA helicase activities. Most of the genes encoding such proteins have been isolated from yeast, on which we will focus in this review. From sequence data, three of the members form a subfamily, the D‐E‐A‐H subfamily.

From unwinding to clamping — the DEAD box RNA helicase family

RNA helicases of the DEAD box family are present in all eukaryotic cells and in many bacteria and Archaea. These highly conserved enzymes are required for RNA metabolism from transcription to degradation and are therefore important players in gene expression. DEAD box proteins use ATP to unwind short duplex RNA in an unusual fashion and remodel RNA–protein complexes, but they can also function as ATP-dependent RNA clamps to provide nucleation centres that establish larger RNA–protein complexes. Structural, mechanistic and molecular biological studies have started to reveal how these conserved proteins can perform such diverse functions and how accessory proteins have a central role in their regulation.

Protein trans-Acting Factors Involved in Ribosome Biogenesis in Saccharomyces cerevisiae

The synthesis of ribosomes is one of the major cellular activities, and in eukaryotes, it takes place primarily, although not exclusively, in a specialized subnuclear compartment termed the nucleolus (125, 155). There, the rRNA genes are transcribed as precursors (pre-rRNAs), which undergo processing and covalent modification. Maturation of pre-rRNAs is intimately linked to their assembly with the ribosomal proteins (r-proteins). These processes depend on various cis-acting elements (6, 188), and they require a large number of nonribosomal protein trans-acting factors (97, 174, 193). Experimental evidence suggests that the basic outline of ribosome synthesis is conserved throughout eukaryotes. However, most of our knowledge comes from the combination of molecular genetic and biochemical approaches in the yeast Saccharomyces cerevisiae. This minireview is aimed at giving an insight into the functions of the many protein trans-acting factors involved in ribosome biogenesis in S. cerevisiae.

Dead-box proteins: a family affair—active and passive players in RNP-remodeling

DEAD-box proteins are characterized by nine conserved motifs. According to these criteria, several hundreds of these proteins can be identified in databases. Many different DEAD-box proteins can be found in eukaryotes, whereas prokaryotes have small numbers of different DEAD-box proteins. DEAD-box proteins play important roles in RNA metabolism, and they are very specific and cannot mutually be replaced. In vitro, many DEAD-box proteins have been shown to have RNA-dependent ATPase and ATP-dependent RNA helicase activities. From the genetic and biochemical data obtained mainly in yeast, it has become clear that these proteins play important roles in remodeling RNP complexes in a temporally controlled fashion. Here, I shall give a general overview of the DEAD-box protein family.

The Q Motif: A Newly Identified Motif in DEAD Box Helicases May Regulate ATP Binding and Hydrolysis

SF1 and SF2 helicases have structurally conserved cores containing seven to eight distinctive motifs and variable amino- and carboxyl-terminal flanking sequences. We have discovered a motif upstream of motif I that is unique to and characteristic of the DEAD box family of RNA helicases. It consists of a 9 amino acid sequence containing an invariant glutamine. A conserved phenylalanine occurs 17 aa further upstream. Sequence alignments, site-specific mutagenesis, and ATPase assays show that this motif and the upstream phenylalanine are highly conserved, that they are essential for viability in the yeast Saccharomyces cerevisiae, and that they control ATP binding and hydrolysis in the yeast translation-initiation factor eIF4A. These results are consistent with computer studies of the solved crystal structures.

The ADE2 gene from Saccharomyces cerevisiae: sequence and new vectors.

We have determined the sequence of a DNA fragment encoding the ADE2 gene from Saccharomyces cerevisiae. A DNA fragment of 2241 bp capable of complementing ade2 mutations was modified so it is available as a single BglII fragment for use in yeast vectors or for gene disruptions. The minimal fragment codes for a putative protein which is highly similar to the protein encoded by the ADE6 gene from Schizosaccharomyces pombe and to the proteins encoded by the purEK operon of Escherichia coli.

Fal1p is an essential DEAD-box protein involved in 40S-ribosomal-subunit biogenesis in Saccharomyces cerevisiae.

A previously uncharacterized Saccharomyces cerevisiae gene, FAL1, was found by sequence comparison as a homolog of the eukaryotic translation initiation factor 4A (eIF4A). Fal1p has 55% identity and 73% similarity on the amino acid level to yeast eIF4A, the prototype of ATP-dependent RNA helicases of the DEAD-box protein family. Although clearly grouped in the eIF4A subfamily, the essential Fal1p displays a different subcellular function and localization. An HA epitope-tagged Fal1p is localized predominantly in the nucleolus. Polysome analyses in a temperature-sensitive fal1-1 mutant and a Fal1p-depleted strain reveal a decrease in the number of 40S ribosomal subunits. Furthermore, these strains are hypersensitive to the aminoglycoside antibiotics paromomycin and neomycin. Pulse-chase labeling of pre-rRNA and steady-state-level analysis of pre-rRNAs and mature rRNAs by Northern hybridization and primer extension in the Fal1p-depleted strain show that Fal1p is required for pre-rRNA processing at sites A0, A1, and A2. Consequently, depletion of Fal1p leads to decreased 18S rRNA levels and to an overall deficit in 40S ribosomal subunits. Together, these results implicate Fal1p in the 18S rRNA maturation pathway rather than in translation initiation.