Production of the small ribosomal subunit in eukaryotes involves the co‐transcriptional assembly of an initial pre‐ribosomal particle containing a subset of ribosomal proteins (RPs) as well as assembly and maturation factors (AFs). This initial pre‐ribosomal particle is split into pre‐40S and pre‐60S pre‐ribosomal particles that follow independent maturation pathways. This review deals with pre‐40S particle maturation only. The nuclear pre‐40S particles are exported via the nuclear pores into the cytoplasm. There, the AFs that function in RP recruitment and control ribosomal RNA (rRNA) folding are sequentially removed. In addition, in yeast at least, cytoplasmic pre‐40S particles are submitted to a quality control process involving a transient interaction with a 60S ribosomal subunit. At the end of the maturation pathway, 18S rRNA is released by an endonucleolytic cleavage.

Newly transcribed RNAs can be metabolically labeled with 4‐thiouridine (s4U) to study many aspects of RNA metabolism genome‐wide, including RNA turnover, transient transcription, and polymerase elongation. New RNAs can be detected via biochemical enrichment (top) or nucleoside recoding to induce U‐to‐C mutations in s4U‐RNA in high‐throughput sequencing.

Enzymes that hydrolyse the 5′ end of RNAs belong to four different enzyme classes: Nudix hydrolases, histidine triad (HIT) proteins, DXO proteins, and ApaH‐like phosphatases.

5‐methylcytosine is emerging as an important epitranscriptomic mark of RNA. Various RNA methyltransferases that reside at different locations in the cell install this mark on a variety of RNA types. These range from cytoplasmic and mitochondrial rRNA and tRNA to mRNA and to various other non‐coding RNAs. Similar to the mulitude of targets, many if not most aspects of RNA metabolism may be affected by the m5C mark.

Transcriptional factors (TFs) regulate mRNA synthesis whereas RNA‐binding proteins (RBPs) regulate mRNA fate, such as stability. Various genome‐wide approaches provide the information for analyzing the function of the gene expression regulators. From these genome‐wide data, computational analysis constructs gene regulatory networks.

Bacterial toxin–antitoxin systems use various mechanisms to inhibit translation and arrest growth by targeting tRNAs.

The magnitude of alternative RNA processing varies across environmental contexts or cellular conditions, with different regulatory modes underlying the severity of changes.