riboWaltz: optimization of ribosome P-site positioning in ribosome profiling data

ABSTRACTRibosome profiling is a powerful technique used to study translation at the genome-wide level, generating unique information concerning ribosome positions along RNAs. Optimal localization of ribosomes requires the proper identification of the ribosome P-site in each ribosome protected fragment, a crucial step to determine trinucleotide periodicity of translating ribosomes, and draw correct conclusions concerning where ribosomes are located. To determine the P-site within ribosome footprints at nucleotide resolution, the precise estimation of its offset with respect to the protected fragment is necessary. Here we present riboWaltz, an R package for calculation of optimal P-site offsets, diagnostic analysis and visual inspection of data. Compared to existing tools, riboWaltz shows improved accuracies for P-site estimation and neat ribosome positioning in multiple case studies.Availability and ImplementationriboWaltz was implemented in R and is available at https://github.com/LabTranslationalArchitectomics/[email protected] or [email protected]

Twin arginine translocation (Tat)-dependent protein transport: the passenger protein participates in the initial membrane binding step

The initial step in twin arginine translocation (Tat)-dependent thylakoid transport of the 16/23 chimera is the interaction of the protein with the lipid bilayer. It results in the formation of the early translocation intermediate Ti-1, which is represented by a protease-protected fragment of 14 kDa. Cys-scanning mutagenesis in combination with in thylakoido and liposome insertion assays was used to precisely map this membrane-interacting and protease-protected fragment within the 16/23 chimera. The fragment comprises 124 residues, which are provided both by the transit peptide (31 residues) and the mature protein (93 residues), demonstrating that the passenger protein directly participates in membrane binding. The implications of this finding on the mechanism of Tat-dependent protein transport are discussed.

Feedback Inhibition of Poly(A)-binding Protein mRNA Translation

An adenine-richciselement at the 5′-untranslated region (UTR) of Pabp1 mRNA is able to inhibit translation of its own mRNA. Similar inhibition of translation of a reporter β-galactosidase mRNA is observed when the adenine-rich auto regulatory sequence (ARS) is placed within the 5′-UTR of this mRNA. For this translational control the distance of the ARS from the 5′ cap is not important. However, it determines the number of 40 S ribosomal subunits bound to the translationally arrested mRNA. Inhibition of mRNA translation by this regulatory sequence occurs at the step of joining of the 60 S ribosomal subunit to the pre-initiation complex. Translational arrest of the ARS containing mRNA in a rabbit reticulocyte lysate cell-free system in the presence of exogenous Pabp1 protects the 5′-flanking region of the ARS from nuclease digestion. This protection depends on the binding of the 40 S ribosomal subunit to the mRNA. The size and the sequence of the nucleotide-protected fragment depends on the location of the ARS within the 5′-UTR. When the ARS is located at a distance of about 78 nucleotides from the 5′ cap, a 40-nucleotide long region adjacent to the ARS is protected. On the other hand, when the ARS is moved further away from the 5′ cap to a distance of ∼267 nucleotides, a 100-nucleotide-long region adjacent to the ARS is protected from nuclease digestion. Nuclease protection is attributed to the presence of one or more stalled 40 S ribosomal subunits near the Pabp1-bound ARS.

Multiple forms of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase are expressed in perinatal rat liver

Fetal rat liver expresses a 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/Fru-2,6-Pase2) form that differs from the adult liver enzyme in the inhibition by phosphorylation by the adenosine 3',5'-cyclic monophosphate-dependent protein kinase and in the recognition by an antibody specific for the NH2-terminal domain of the adult liver enzyme. Northern blot analysis shows that fetal hepatocytes contain a species of mRNA that is 2.2 kb in size and that exhibits the maximal levels after delivery. PFK-2/Fru-2,6-Pase2 mRNA analysis using a sensitive ribonuclease protection assay reveals the presence of nearly similar amounts of adult liver-specific and skeletal muscle-specific mRNA in fetal liver and hepatocytes during the last days of gestation, as well as a 233-bp protected fragment present in fetal liver. These results were confirmed by polymerase chain reaction using specific oligonucleotide pairs. Primer extension of fetal liver cDNA suggests the presence of two initiation sites of transcription. Analysis of the adult liver PFK-2/Fru-2,6-Pase2 protein during the perinatal transition using a specific antibody shows a marked accumulation of this form immediately after birth.

Genes required for completion of import of proteins into the endoplasmic reticulum in yeast.

Yeast secretory mutants sec53 and sec59 define a posttranslational stage in the penetration of glycoprotein precursors into the endoplasmic reticulum (ER). In the previous report we showed that at the restrictive temperature (37 degrees C) these mutants accumulate enzymatically inactive and incompletely glycosylated forms of the secretory enzyme invertase and the vacuolar enzyme carboxypeptidase Y. Cell fractionation experiments reveal that these precursor forms remain firmly bound to the ER membrane. However, upon return to the permissive temperature (24 degrees C), the invertase precursors are glycosylated, become partially active, and are secreted. Thermoreversible conversion does not require protein synthesis, but does require energy. In contrast to the effect of these mutations, inhibition of oligosaccharide synthesis with tunicamycin at 37 degrees C causes irreversible accumulation of unglycosylated invertase. The effect of the drug is exaggerated by high temperature since unglycosylated invertase synthesized in the presence of tunicamycin at 25 degrees C is secreted. A portion of the invertase polypeptide accumulated at 37 degrees C is preserved when membranes from sec53 and sec59 are treated with trypsin. In the presence of Triton X-100 or saponin, the invertase is degraded completely. The protected fragment appears to represent a portion of the invertase polypeptide that is embedded in or firmly associated with the ER membrane. This association may develop early during the synthesis of invertase, so that in the absence of translocation, some of the completed polypeptide chain remains exposed on the cytoplasmic surface of the ER.