David Boulware and colleagues investigate clinical features in a prospective cohort with AIDS and recent cryptococcal meningitis after initiation of antiretroviral therapy to identify biomarkers for prediction and diagnosis of CM-IRIS (cryptococcal meninigitis-related immune reconstitution inflammatory syndrome).

The function of the U3 small nucleolar ribonucleoprotein (snoRNP) is central to the events surrounding prerRNA processing, as evidenced by the severe defects in cleavage of pre-18S rRNA precursors observed upon depletion of the U3 RNA and its unique protein components. Although the precise function of each component remains unclear, since U3 snoRNA levels remain unchanged upon genetic depletion of these proteins, it is likely that the proteins themselves have significant roles in the cleavage reactions. Here we report the identification of two previously undescribed protein components of the U3 snoRNP, representing the first snoRNP components identified by using the two-hybrid methodology. By screening for proteins that physically associate with the U3 snoRNP-specific protein, Mpp10p, we have identified Imp3p (22 kDa) and Imp4p (34 kDa) (named for interacting with Mpp10p). The genes encoding both proteins are essential in yeast. Genetic depletion reveals that both proteins are critical for U3 snoRNP function in pre-18S rRNA processing at the A0, A1, and A2 sites in the pre-rRNA. Both Imp proteins associate with Mpp10p in vivo, and both are complexed only with the U3 snoRNA. Conservation of RNA binding domains between Imp3p and the S4 family of ribosomal proteins suggests that it might associate with RNA directly. However, as with other U3 snoRNP-specific proteins, neither Imp3p nor Imp4p is required for maintenance of U3 snoRNA integrity. Imp3p and Imp4p are therefore novel protein components specific to the U3 snoRNP with critical roles in pre-rRNA cleavage events.Eukaryotic ribosomes are large ribonucleoproteins (RNPs) composed of four rRNAs and dozens of ribosomal proteins. Three of the four RNA components are processed from a single polycistronic transcript, undergo extensive nucleotide modification, and assemble with the appropriate proteins in the cell nucleolus. In the yeast Saccharomyces cerevisiae, the small-subunit (SSU) rRNA (18S) and the large-subunit (LSU) rRNAs (5.8S and 25S) are processed from the nascent 35S transcript via a series of cleavage steps (Fig. 1A). Successive cleavages at the 5Ј end of the precursor rRNA yield the 33S and 32S transcripts, while subsequent cleavage at the A2 site separates the precursors of the SSU rRNA from those of the LSU rRNA. The 20S pre-rRNA is matured to 18S rRNA, while the LSU precursors are terminally processed to the 5.8S and 25S rRNAs.Several small nucleolar RNPs (snoRNPs) are required for the cleavage steps that generate the mature 18S rRNA (reviewed in reference 35). Of these, the most widely studied is the U3 snoRNP. In yeast, genetic depletion of the U3 snoRNA results in a deficiency in processing at the A0, A1, and A2 sites, causing a sharp decrease in 20S and 18S rRNA levels and an apparent increase in the presence of the 23S precursor and the 35S nascent transcript (Fig. 1B). A role for the U3 snoRNP in pre-rRNA cleavage events has also been observed in metazoans. Oligonucleotide-mediated depletion of the U3 snoRNA in vivo in Xenopus laevis oocytes an...

BACKGROUND:Th e surviving sepsis guidelines recommend early aggressive fl uid resuscitation within 6 h of sepsis onset. Although rapid fl uid administration may off er benefi t, studies on the timing of resuscitation are lacking. We hypothesized that there is an association between quicker, adequate fl uid resuscitation and patient outcome from sepsis onset time.

The Saccharomyces cerevisiae Rlp7 protein has extensive identity and similarity to the large ribosomal subunit L7 proteins and shares an RNA-binding domain with them. Rlp7p is not a ribosomal protein; however, it is encoded by an essential gene and therefore must perform a function essential for cell growth. In this report, we show that Rlp7p is a nucleolar protein that plays a critical role in processing of precursors to the large ribosomal subunit RNAs. Pulse-chase labeling experiments with Rlp7p-depleted cells reveal that neither 5.8S S, 5.8SL, nor 25S is produced, indicating that both the major and minor processing pathways are affected. Analysis of processing intermediates by primer extension indicates that Rlp7p-depleted cells accumulate the 27SA 3 precursor RNA, which is normally the major substrate (85%) used to produce the 5.8S and 25S rRNAs, and the ratio of 27SB L to 27SBS precursors changes from approximately 1:8 to 8:1 (depleted cells). Because 27SA 3 is the direct precursor to 27SBS, we conclude that Rlp7p is specifically required for the 5 to 3 exonucleolytic trimming of the 27SA 3 into the 27SBS precursor. As it is essential for processing in both the major and minor pathways, we propose that Rlp7p may act as a specificity factor that binds precursor rRNAs and tethers the enzymes that carry out the early 5 to 3 exonucleolytic reactions that generate the mature rRNAs. Rlp7p may also be required for the endonucleolytic cleavage in internal transcribed spacer 2 that separates the 5.8S rRNA from the 25S rRNA. Ribosomes are large ribonucleoproteins that translate mRNA into protein. They are ribozymes, possessing solely RNA in their active site (1). The large and small ribosomal subunits harbor distinct RNA and protein components. In the yeast Saccharomyces cerevisiae, the small subunit bears only 1 rRNA, the 18S, whereas the large subunit bears 3 rRNAs, the 5S, 5.8S, and 25S. The 18S, 5.8S, and 25S rRNAs are processed from a polycistronic RNA polymerase I transcript and assembled with their respective small and large subunit proteins in the cell nucleolus. Ribosome biogenesis is highly regulated during the cell cycle and under different growth conditions (2). An insufficient number of ribosomes may be a checkpoint for cell growth in size and therefore for cell cycle progression (3).Ribosome biogenesis in S. cerevisiae begins with transcription of a 35S precursor rRNA (pre-rRNA) transcript that is modified at the nucleotide level and processed at a number of specific sites to release the mature 18S, 5.8S, and 25S rRNAs ( Fig. 1; refs. 4, 5). Processing of RNA precursors to the 18S rRNA requires a number of endonucleolytic cleavage reactions. The four small nucleolar ribonucleoproteins (snoRNPs), U3, U14, snR10, and snR30 play an essential role in these reactions, but none of them have yet been shown to possess the cleaving enzyme. Processing of the large subunit RNAs, 5.8S and 25S, requires both endonucleolytic cleavage and exonucleolytic processing of the cleavage products and begins on the 27SA 2 pre...

The U3 small nucleolar ribonucleoprotein (snoRNP) is required for three cleavage events that generate the mature 18S rRNA from the pre-rRNA. In Saccharomyces cerevisiae, depletion of Mpp10, a U3 snoRNP-specific protein, halts 18S rRNA production and impairs cleavage at the three U3 snoRNP-dependent sites: A0, A1, and A2. We have identified truncation mutations of Mpp10 that affect 18S rRNA synthesis and confer cold-sensitivity and slow growth. However, distinct from yeast cells depleted of Mpp10, the mutants carrying these truncated Mpp10 proteins accumulate a novel precursor, resulting from cleavage at only A0. The Mpp10 truncations do not alter association of Mpp10 with the U3 snoRNA, nor do they affect snoRNA or protein stability. Thus, the role in processing of the U3 snoRNP can be separated into cleavage at the A0 site, which occurs in the presence of truncated Mpp10, and cleavage at the A1͞A2 sites, which occurs only with intact Mpp10. These results strongly argue for a role for Mpp10 in processing at the A1͞A2 sites.Four rRNAs and many proteins comprise the eukaryotic ribosome: the 60S subunit contains 25S (Saccharomyces cerevisiae), 5.8S, and 5S rRNAs, and the 40S subunit houses 18S rRNA. In the nucleolus RNA polymerase I transcribes long, precursor molecules that must be processed to yield the mature 25S, 5.8S, and 18S rRNAs. A schematic of pre-rRNA processing (Fig. 1) shows that the rRNA sequences are flanked by two external transcribed spacers (5Ј and 3Ј) and separated by two internal transcribed spacers (ITS1 and ITS2). Complexes of a small nucleolar RNA and proteins, called snoRNPs, and other nucleolar proteins process this original transcript into the mature rRNAs. The depletion or mutation of many of these processing molecules disrupts pre-rRNA processing (1, 2).Many small nucleolar RNAs (snoRNAs) have been identified but U3 is the most abundant in vertebrate cells and exists in a wide evolutionary range of species (from trypanosomes to humans), making it an attractive molecule for studies aimed at elucidation of pre-rRNA processing. In vivo and͞or in vitro studies in three different systems, mouse, Xenopus laevis oocytes, and S. cerevisiae, have demonstrated that it is as an essential component for production of 18S rRNA (3-9).In yeast, processing of the pre-18S rRNA occurs before the cleavage steps that release the mature 5.8S and 25S rRNAs (Fig.