The majority of genes carried in a cell’s DNA specify the amino acid sequence of proteins; the RNA molecules

that are
copied from these genes (which ultimately direct the synthesis of proteins) are called messenger RNA (mRNA) molecules.

The final product of other genes, however, is the RNA molecule itself. These RNAs are known as noncoding RNAs because
they do not code for protein.

In a well-studied, single-celled eukaryote, the yeast Saccharomyces cerevisiae, over 1200 genes (more than 15% of the total)
produce RNA as their final product. Humans may produce on the order of ten thousand noncoding RNAs. These RNAs, like
proteins, serve as enzymatic, structural, and regulatory components for a wide variety of processes in the cell.
Eukaryotes solve the problem of replicating the ends of their linear chromosomes with a specialized end structure, the telomere,
maintained by a special nucleotide polymerizing enzyme called telomerase. Telomerase extends one of the DNA strands at the end of a
chromosome by using an RNA template that is an integral part of the enzyme itself, producing a highly repeated DNA sequence that
typically extends for thousands of nucleotide pairs at each chromosome end. Telomeres have specialized structures that distinguish them
from broken ends of chromosomes, ensuring that they are not mistakenly repaired.
RNA Splicing Is Performed by the Spliceosome

Unlike the other steps of mRNA production, we have discussed, key steps in RNA splicing are performed by RNA
molecules rather than proteins. Specialized RNA molecules recognize the nucleotide sequences that specify where
splicing is to occur and also catalyze the chemistry of splicing. These RNA molecules are relatively short (less than
200 nucleotides each), and there are five of them, U1, U2, U4, U5, and U6. Known as snRNAs (small nuclear
RNAs), each is complexed with at least seven protein subunits to form an snRNP (small nuclear ribonucleoprotein).

These snRNPs form the core of the spliceosome, the large assembly of RNA and protein molecules that performs
pre-mRNA splicing in the cell. During the splicing reaction, recognition of the 5ʹ splice junction, the branch-point
site, and the 3ʹ splice junction is performed largely through base-pairing between the snRNAs and the consensus
RNA sequences in the pre-mRNA substrate. The spliceosome is a complex and dynamic machine. When studied in
vitro, a few components of the spliceosome assemble on pre-mRNA and, as the splicing reaction proceeds, new
components enter and those that have already performed their tasks are jettisoned (Figure 6–28). However, many
scientists believe that, inside the cell, the spliceosome is a preexisting, loose assembly of all the components—
capturing, splicing, and releasing RNA as a coordinated unit, and undergoing extensive rearrangements each time a
splice is made.
Figure 6–27: The consensus nucleotide sequences in an RNA molecule that signal the beginning and the end of most introns in
humans. The three blocks of nucleotide sequences shown are required to remove an intron sequence. Here A, G, U, and C are
the standard RNA nucleotides; R stands for purines (A or G); and Y stands for pyrimidines (C or U). The A highlighted in red
forms the branch point of the lariat produced by splicing (see Figure 6–25).

Only the GU at the start of the intron and the AG at its end are invariant nucleotides in the splicing consensus sequences.
Several different nucleotides can occupy the remaining positions, although the indicated nucleotides are preferred. The
distances along the RNA between the three splicing consensus sequences are highly variable; however, the distance between the
branch point and 3ʹ splice junction is typically much shorter than that between the 5ʹ splice junction and the branch point.
There are four types of eukaryotic rRNAs, each present in one copy per ribosome. Three of the four rRNAs (18S, 5.8S, and 28S) are made by
chemically modifying and cleaving a single large precursor rRNA (Figure 6–40); the fourth (5S RNA) is synthesized from a separate cluster of
genes by a different polymerase, RNA polymerase III, and does not require chemical modification.

Extensive chemical modifications occur in the 13,000-nucleotide-

long precursor rRNA before the rRNAs are cleaved out of it and
assembled into ribosomes. These include about 100 methylations
of the 2ʹ-OH positions on nucleotide sugars and 100
isomerizations of uridine nucleotides to pseudouridine (Figure 6–
41A). The functions of these modifications are not understood in
detail, but they probably aid in the folding and assembly of the
final rRNAs, or subtly alter the function of ribosomes. Each
modification is made at a specific position in the precursor rRNA,
specified by “guide RNAs,” which position themselves on the
precursor rRNA through base-pairing and thereby bring an RNA-
modifying enzyme to the appropriate position (Figure 6–41B).
Other guide RNAs promote cleavage of the precursor rRNAs into
the mature rRNAs, probably by causing conformational changes
in the precursor rRNA that expose these sites to nucleases. All of
these guide RNAs are members of a large class of RNAs called
small nucleolar RNAs (or snoRNAs), so named because these
RNAs perform their functions in a subcompartment of the nucleus
called the nucleolus. Many snoRNAs are encoded in the introns of
other genes, especially those encoding ribosomal proteins. They
are synthesized by RNA polymerase II and processed from
excised intron sequences.
 The Nucleolus Is a Ribosome-Producing Factory

Ribosome assembly is a complex process, the most

important features of which are outlined in Figure 6–45.
In addition to its central role in ribosome biogenesis, the
nucleolus is the site where other noncoding RNAs are
produced and other RNA–protein complexes are
assembled. For example, the U6 snRNP, which functions
in pre-mRNA splicing (see Figure 6–28), is composed of
one RNA molecule and at least seven proteins. The U6
snRNA is chemically modified by snoRNAs in the
nucleolus before its final assembly there into the U6
snRNP. Other important RNA–protein complexes,
including telomerase (encountered in Chapter 5) and the
signal-recognition particle (which we discuss in Chapter
12), are assembled at the nucleolus. Finally, the tRNAs
(transfer RNAs) that carry the amino acids for protein
synthesis are processed there as well; like the rRNA
genes, the genes encoding tRNAs are clustered in the
nucleolus. Thus, the nucleolus can be thought of as a
large factory at which different noncoding RNAs are
transcribed, processed, and assembled with proteins to
form a large variety of ribonucleoprotein complexes.
Group I introns are large self-splicing ribozymes. They catalyze their own excision from mRNA, tRNA and rRNA precursors
in a wide range of [Link] core secondary structure consists of nine paired regions (P1-P9). These fold to essentially
two domains– the P4-P6 domain (formed from the stacking of P5, P4, P6 and P6a helices) and the P3-P9 domain (formed
from the P8, P3, P7 and P9 helices.
Splicing of group I introns is processed by
two
sequential transesterification reactions. First
an exogenous guanosine or
guanosine nucleotide (exoG) docks onto the
active G-binding site located in P7, and then
its 3'-OH is aligned to attack
the phosphodiester bond at the "upstream"
(closer to the 5' end) splice site located in
P1, resulting in a free 3'-OH group at the
upstream exon and the exoG being attached
to the 5' end of the intron. Then the terminal
G (omega G) of the intron swaps out the
exoG and occupies the G-binding site,
preparing the second ester-transfer reaction:
the 3'-OH group of the upstream exon in P1
is aligned to attack the downstream splice
site in P10, leading to the ligation of the
adjacent upstream and downstream exons
and release of the catalytic intron.
Piwi-interacting RNA (piRNA) is the largest class of small non-coding RNA molecules expressed in animal cells.
piRNAs form RNA-protein complexes through interactions with piwi-subfamily Argonaute proteins. These piRNA
complexes are mostly involved in the epigenetic and post-transcriptional silencing of transposable elements.

The length of a piRNA varies between species (from 21 to 31 nucleotides) and piRNA have a 5’ monophosphate and a
3’ uridine in which 2’ or 3’ oxygen is modified by methylation. The 2’-O-methylation has been suggested that it
increases piRNA stability.

More than 50,000 unique piRNA sequences have been discovered in mice and more than 13,000 in D.
melanogaster. It is thought that there are many hundreds of thousands of different piRNA species in mammals.
(A) A model of the primary piRNA biogenesis pathway.
The piRNA precursors are transcribed from piRNA
clusters, and are then processed into piRNA
intermediates. The piRNA intermediates with uridine at
the 5’ ends are loaded onto Piwi proteins, with HSP90
facilitates the loading. Subsequently, the 3’ portions of
piRNA intermediates are trimmed by unidentified
nuclease(s). After the trimming, 3’ ends are 2’-O-
methylated by Hen1 methyltransferase. Mitochondrial
outer membrane proteins MitoPLD/Zucchini, GASZ,
and GPAT2/Minotaur are probably involved in the
processing of piRNA precursors or intermediates

(B) A model of the secondary biogenesis pathway. The

Piwi/piRNA complexe cleaves a transposon RNA
between the 10th and 11th position of piRNAs. The 3’
region of the cleaved RNA is incorporated into Piwi
proteins. The 5’ region is ejected from Piwi proteins by
chaperone machinery FKBP6/Shutdown and HSP90, and
is then degraded. The 10th position of the incorporated
RNA is enriched in adenine, because it is
complementary to the first position of a piRNA that is
enriched in uridine. The incorporated RNA is then
processed into a mature secondary piRNA by trimming
and modification likely by the same mechanisms that
generate a primary piRNA.
The secondary pathway, also known as the ping-pong cycle,
selectively amplifies specific piRNAs targeting active
transposons (see previous figure and the Figure on the right).
Of the three fly Piwi proteins, Aubergine and AGO3 are
involved in this process. Aubergine binds to piRNAs generated
from both primary (primary piRNAs) and secondary pathways
(secondary piRNAs), while AGO3 binds only to secondary
piRNAs. The secondary pathway begins with cleavage of sense
transposon transcripts by Aubergine bound to primary piRNAs
derived from antisense transposon sequences

Function: Like other small RNAs, piRNAs are thought to be involved in gene
silencing, specifically the silencing of [Link] mammals, it appears that the
activity of piRNAs in transposon silencing is most important during the development
of the embryo and in both C. elegans and humans, piRNAs are necessary
for spermatogenesis.
The Pc-G proteins are not conventional
repressors. They are not responsible for
deter mining the initial pattern of
expression of the genes on which they act.
In the absence of Pc G proteins, these genes
are initially repressed as usual, but later in
development the repres sion is lost without
Pc-G group functions. This suggests that
the Pc-G proteins in some way rec1g nize
the state of repression when itis established,
and they then act to perpetuate it thrlugh
cell division of the daughter cells. Figure
31.9 shows a model in which Pc-G proteins
bind in conjunction with a repressor, but the
Pc-G proteins remain bound after the
repressor is no longer available. This is
necessary to maintain repression, so that if
Pc-G proteins are absent, the gene becomes
activated.
PRC2 (polycomb repressive complex 2) is one of the two classes of polycomb-group
proteins or (PcG). The other component of this group of proteins is PRC1 (Polycomb
Repressive Complex 1).
This complex has histone methyltransferase activity and primarily methylates histone
H3 on lysine 27 (i.e. H3K27me3) a mark of transcriptionally silent chromatin. PRC2 is
required for initial targeting of genomic region (PRC Response Elements or PRE) to be
silenced, while PRC1 is required for stabilizing this silencing and underlies cellular
memory of silenced region after cellular differentiation.

PRC1 also mono-ubiquitinates histone H2A on lysine 119 (H2AK119Ub1).

These proteins are required for long term epigenetic silencing of chromatin and have an
important role in stem cell differentiation and early embryonic development.